JETIR Research Journal

Identification and characterization of a naturally occurring agglutinin in the hemolymph of the marine crab, Atergatis latissimus (H. Milne Edwards, 1834)

Elayabharathi, T1, Vinoliya Josephine Mary , J, Mary Mettilda Bai, S3 1Research Scholar (MSU/RES/R1/Reg. No. 10165), 2Assistant professor, 3Assistant professor 1,2,3 Department of Zoology, Holy Cross College (Autonomous), Nagercoil. 1Affiliated to Manonmaniam Sundaranar University, Abishekapatti, Tirunelveli - 627012, Tamil Nadu, India.

Abstract : A naturally occurring hemagglutinin with strong affinity for buffalo erythrocytes was detected in the hemolymph of the marine crab, Atergatis latissimus. Hemagglutination activity was stable between pH 7 and 9.5 and temperature from 0°C to 30°C suggesting the agglutinin to be pH and temperature sensitive. Addition of divalent cations (Ca2+, Mg2+ and Mn2+) increased the HA titre up to 5.0 mM and decreased with increase in concentration. Atergatis latissimus agglutinin exhibited an increase in HA titre with trypsin and neutral protease treated rabbit erythrocytes and neuraminidase treatment reduced the HA when compared to native erythrocytes. The heamagglutinability of the agglutinin was inhibited by glycoproteins: BSM > bovine thyroglobulin > fetuin = PSM > transferrin > apo- transferrin = lactoferrin and sugars raffinose > trehalose = α - Lactose = melibiose. Disappearance of agglutinability following cross adsorption revealed the presence of a single agglutinin. Thus the preliminary characterization of the hemolymph agglutinin would provide strategies for purification of a lectin from the marine crab, Atergatis latissimus.

Key words: Agglutinin, Glycocalyx, Hemagglutination, Hemagglutination inhibition, Sialic acid. ______

I. INTRODUCTION The defense system of invertebrates relies on innate immune mechanisms to protect themselves against various infectious pathogens (Ghosh et al., 2011) and is essential for the survival of all organisms (Salzet, 2001). Pathogens are recognized by cell derived pattern recognition receptors with diverse binding specificity (Smith, 2016) and biologically active molecules that occur naturally or induced and help in the elimination of non-self. Among the humoral molecules, agglutinins/lectins which are sugar binding proteins are best studied owing to their multiple binding sites and specificity to bind to either the whole sugar or to a specific site / sequence of a sugar or their glycosidic linkages on cell surface glycol-conjugates, or in bacterial polysaccharides (Krishnamoorthi et al., 2016; Gasmi et al., 2017). They can bind to sugar moieties in cell walls or membranes and thereby change the physiology of the membrane to cause agglutination or other biochemical changes in the cell (Hamid et al., 2013; Sullivan, 2017). Lectins recognize the normal and pathogenic cells by interaction with the carbohydrate moieties expressed on the cell surface (Kovar et al., 2000). They are structurally diverse composed of subunits varying in molecular size and metal requirements (Sharon, 2008) and can agglutinate a variety of animal cells by binding to cell surface glycoproteins and glycolipids (Sharon, 2008). The specificity and avidity of lectin-carbohydrate interaction depends on the structure of the terminal monosaccharide residue, configuration of the glycosidic bond between monomers and branching degree of a glycan (Neth et al., 2000). Agglutinating activity depends on the nature, number distribution, exposure of cell surface receptors and surface charges of the membrane (Lis and Sharon, 1986). Physico-chemical factors like pH which determines the ionization state and temperature - the thermal tolerance of the agglutinin (Reeves and Rahn, 1979), influences the binding affinity of the lectin with the interacting sugar. Marine lectins are identified by their metal ion requirement as C-type lectins because of the affinity to calcium ions (Gowda et al., 2008a) and are required for binding to their carbohydrate protein domains. Removal of calcium reduces the agglutination ability and it is confirmed by the addition of calcium chelators (Philip et al., 2013). C-type lectins have been documented from marine crabs and are known to be sialic acid specific (Na et al., 2007). Sialic acid specific lectins play a major role in discriminating normal and pathogenic cells and in clearance of pathogens from the system of invertebrates. Thus isolation of a sialic acid specific lectin requires characterization of lectin. Hence the present study was undertaken to partially characterize the agglutinin from the hemolymph of marine crab A. latissimus and identify the specific ligand that would help in the purification and therapeutic application of the lectin.

2. MATERIALS AND METHODS

2.1. Animal collection and maintenance: The crabs, Atergatis latissimus were collected from the coastal areas of Arockyapuram, Kanyakumari District, Tamilnadu, India. They were maintained in plastic containers with sea water, fed with anchovy fish and the water was replenished daily. 2.2. Collection and preparation of mammalian erythrocytes: Buffalo, mice, rat, guinea pig, rabbit, pig, dog, Human, A, B, O, camel, cow, goat, horse and donkey erythrocytes were prepared following the standard method of Ravindranath and Paulson (1987).

2.3. Collection of hemolymph: The hemolymph from the crab Atergatis latissimus was collected following the procedure of Mercy and Ravindranath (1992).

2.4. Hemagglutination (HA) and Hemagglutination inhibition (HAI) Assay Hemagglutination assay and Hemagglutination inhibition assay were performed in 96 well, „U` bottomed microtiter plates (Tarson) as described by Ravindranath and Paulson (1987).

JETIR1810299 Journal of Emerging Technologies and Innovative Research (JETIR) www.jetir.org 593

2.5. pH and thermal stability : pH and temperature dependence of the agglutinin was tested by pre incubating the hemolymph at different pH (5-10) and temperature (0oC - 100oC) for 1 hour before adding erythrocyte suspension and was checked for HA activity. 2.6. Cation dependency and EDTA sensitivity: To assess the effect of cations and EDTA on the HA activity, 25 µl of hemolymph was 2+ 2+ 2+ serially diluted with equal volume of TBS of different concentrations (0 -100 mM) of divalent cations (Ca , Mg and Mn ) and calcium chelators (EDTA and trisodium citrate). After incubation, the HA activity of each sample was determined. 2.7. Trypsin and protease treatment: Trypsin and protease treatment was carried out following the procedure of Pereira et al. (1981). 2.8. Neuraminidase treatment: Asialo erythrocytes were prepared following the method of Mercy and Ravindranath (1993). The desialylated erythrocyte were used for hemagglutination assay.

2.9. Cross adsorption assay: The cross adsorption assay was carried out following the method of Mercy and Ravindranath (1992).

3. RESULTS

3.1. Hemagglutination assay Hemolymph agglutinin of the marine crab, Atergatis latissimus agglutinated the mammalian erythrocytes as follows: dog > buffalo > mice > rat > rabbit = guinea pig = human A=B=0 > camel = goat = pig = horse. Maximum agglutination was observed with dog followed by buffalo erythrocytes (Table -1). Since the HA titre fluctuated with different dog erythrocytes, further characterization was carried out with buffalo erythrocytes.

Table-1: Hemagglutination titer of hemolymph agglutinin of Atergatis latissimus with different mammalian erythrocytes

Erythrocytes (n=10) HA titer Dog 1024 Buffalo 512 Mice 128 Rat 32 Rabbit 16 Guinea pig 16 Human A 16 Human B 16 Human O 16 Camel 8 Goat 8 Pig 8 Horse 8 Donkey 8 Cow 4

3.2. Influence of pH , temperature, divalent cation and calcium chelator on HA The hemolymph agglutinin was sensitive to changes in pH and temperature. The hemagglutinating activity was stable from pH 7 to 9.5 and remained unaffected by change in the temperature from 0°C-30°C (Table -2). Divalent cations (Ca2+, Mg2+ and Mn2+) decreased the agglutination of the hemolymph of the marine crab, A. latissimus at concentration up to 1 mM. With further increase in cation concentration an increase in HA was noted followed by a reduction in HA at higher concentration (Table -3). When the hemagglutinating activity of the hemolymph was tested in the presence of varying concentrations of calcium chelators, a reduction in HA titre was observed with disodium EDTA and tetrasodium EDTA whereas reduction in HA was observed only above 40 mM concentration with trisodium citrate (Table-4).

Table-2: Hemagglutination titer of hemolymph of Atergatis latissimus in relation to change in pH and temperature

pH Temperature oC HA titer HA titer (n=5) (n=5) 5 0 64 512 5.5 10 64 512 6 20 128 512 6.5 30 256 512 7 40 512 128 JETIR1810299 Journal of Emerging Technologies and Innovative Research (JETIR) www.jetir.org 594

7.5 50 512 16 8 60 512 0 8.5 70 512 0 9 80 512 0 9.5 90 512 0 10 100 128 0

Table-3: Effects of cations on the hemaglutinating activity of the agglutinin of the marine crab Atergatis latissimus

Cation conc. in mM HA titer (n=10) Ca2+ Mg2+ Mn2+ 0 256 256 256 0.01 256 256 256 0.1 256 256 256 1.0 512 256 256 5.0 512 512 512 10 512 512 512 20 512 512 256 30 256 256 128 40 256 128 128 50 128 64 64 100 128 32 64

Table- 4: Effect of chelators on the hemagglutinating activity of the naturally occurring agglutinin in the hemolymph of marine crab Atergatis latissimus

Concentration in EDTA mM Trisodium citrate Disodium Tetrasodium (n=10) 0 256 256 256

0.01 256 256 256

0.1 256 256 256

1.0 512 512 256

5.0 512 512 512

10 16 64 512

15 4 32 256

20 4 8 128

30 2 4 64

40 2 4 32

50 0 0 16

100 0 0 8

JETIR1810299 Journal of Emerging Technologies and Innovative Research (JETIR) www.jetir.org 595

3.3. Hemagglutination inhibition (HAI) assay Agglutinability of the agglutinin was inhibited by glycoproteins: Bovine submaxillary mucin > thyroglobulin > fetuin = PSM (Table-5) and sugars: raffinose > Trehalose = α-Lactose = melibiose = L-fucose (Table -6).

Table-5: Hemagglutination inhibition of the hemolymph agglutinin of the marine crab Atergatis latissimus by various glycoproteins Relative inhibitory potency Glycoproteins Minimum conc. HAI (%) (n=5) Required (mM) BSM 256 19.53 100 Bovine Thyroglobulin 64 39.06 50 Fetuin 32 78.25 25 PSM 32 78.125 25 Transferin 16 156.26 12.5 Apo – transferin 16 156.25 12.5 Lactoferrin 8 312.5 6.25

Table- 6: Hemagglutination inhibition (HAI) of the hemolymph agglutinin of the marine crab Atergatis latissimus by various sugars

Sugars Minimum conc. Relative inhibitory potency HAI Required (mM) (%)

Raffinose 32 3.12 100 Trehalose 16 6.25 50 α-Lactose 16 6.25 50 Melibiose 16 6.25 50 L-Fucose 16 6.25 50 GluNAc 8 12.5 25 Man NAc 8 12.5 25 Glu-3-PO4 4 25 12.5 D-glucose 4 25 12.5 Glu-6-PO4 4 25 12.5

3.4. Enzyme treatment on HA Neuraminidase treatment of buffalo erythrocytes tremendously reduced the HA titer, whereas treatment of buffalo erythrocytes with trypsin and neutral protease enhanced the HA titer of the hemolymph agglutinin (Table -7).

Table-7: Effect of enzyme treatment of buffalo erythrocytes on the hemolymph agglutinin of Atergatis latissimus

Enzyme Site of enzyme activity HA titer (n=5) None - 512 Neuraminidase (Clostridium Neu Ac-D-Gal; Neu 32 perfringens Type X) Ac-D-GalNAc Trypsin (1mg/ml) Arg-Lys-S 1024 Neutral protease (1mg/ml) - 1024

3.5. Effect of cross adsorption on HA titer Cross adsorption profile of the hemolymph of A. latissimus indicated the presence of a single agglutinin in the hemolymph as evidenced by the disappearance of the HA activity following adsorption with any erythrocyte that showed agglutination (Table -8).

Table-8: Hemagglutination titer of the hemolymph of marine crab Atergatis latissimus after adsorption with different erythrocytes

Erythrocytes EDTA titer adsorbed Rat Rabbit Buffalo Dog Mice

None 512 1024 128 32 16

JETIR1810299 Journal of Emerging Technologies and Innovative Research (JETIR) www.jetir.org 596

Buffalo 0 0 0 0 0 Dog 2 (0) 0 8 (0) 4 (0) 0 Mice 0 0 0 4 (0) 0 Rat 2 (0) 8 (0) 0 0 0 Rabbit 0 0 0 0 0

4. DISCUSSION

The present study attempted to characterize the physico-chemical conditions required for optimum activity of the lectin identified in the serum of the crab A. latissimus. Among the various mammalian erythrocytes tested against the serum of the crab A. latissimus, dog, buffalo and mice erythrocytes showed high titre value. The specific preference of the hemolymph of crab A. latissimus to buffalo erythrocytes suggests that the receptor determinants of buffalo erythrocytes are specifically recognized by the hemolymph agglutinin. The agglutinin preferentially agglutinated buffalo erythrocytes that contain NeuGc (Chien et al., 1978), dog erythrocyte that is specific to NeuGc/NeuAc (Yasue et al., 1978) and mice erythrocyte with O-acetyl group (Varki and Kornfeld, 1980). So it can be concluded that the agglutinin may bind to the sialic acid of the glycocalyx of these erythrocytes (Yosue et al., 1978). High HA titer was observed in the hemolymph of the marine crab Atergatis latissimus between pH 7-9.5, which got reduced below pH 7 and above pH 9.5. Hauzer et al. (1979) studied the pH dependence of the lectins and found increased agglutination at neutral and slightly alkaline pH which supports our finding. The inactivation of the lectin at acidic and highly alkaline pH may be due to demetallization followed by irreversible conformational changes (Duk et al., 1984). Hemolymph lectin was active at temperature ranging from 0° - 30°C and disappeared completely at 60°C as reported by Scott (1971). Difference in hemagglutination titre with varying temperature may be primarily due to the structural transition of the lectin (Huet, 1975) and loss of biological activity at higher temperatures may be due to the destabilization of weak interactions necessary for the native conformation of the lectin (Singh and Saxena, 2013). Agglutinin with pH and temperature sensitivity is also reported in the hemolymph of Atergatis reticulates (Denis and Mercy, 1999), Atergatis subdentatus (Denis et al., 2016), Atergatis ocyroe (Elayabharathi and Vinoliya, 2017). Divalent cations are necessary for ligand binding and the structure of the lectin is altered by demetallization (Abhilash and Haridas, 2015). Hosoi et al.(1998), observed that presence of excess calcium ion stabilizes the lectin conformation and if calcium concentration is limited sugar binding as well as protein -protein interaction becomes apparent. In the present study significant increase in HA was not observed suggesting that the agglutinin in the hemolymph is rich in endogenous calcium and does not respond to the exogenous cations for its activity. To confirm the lectin as calcium dependent, EDTA a metal chelating agent was added to the lectin. Decrease in HA activity was observed suggesting the lectin as calcium dependent. C-type lectins are reported in crabs, Atergatis subdentatus (Denis et al., 2016) and Travancoriana charu (Sheeja, 2017). Susceptibility of red blood cells to agglutinins generally increases when they are treated with trypsin (Elayabharathi and Vinoliya, 2017). Cell agglutinability may be affected not only by the binding capacity but also by the cell surface topography (Ishikawa et al., 1981). Protease treatment of buffalo erythrocytes may remove the cell surface proteins that exposes most of the agglutinin binding sites, possibly the sugar residues of gangliosides recognized by the agglutinin thus enhancing the HA titre. Hemagglutination inhibition tests performed in this study revealed that saccharides like raffinose > α - lactose = melibiose = trehalose inhibited the agglutinating activity of A. latissimus serum when compared to other sugars. In addition, the hemagglutination activity was inhibited by glycoproteins BSM > thyroglobulin > fetuin > PSM > transferrin. BSM, which contains mainly 9-O-acetyl and 8,9-di-O - acetyl-N-acetyl neuraminic acid (Schaver, 1982), was the most potent inhibitor of the agglutinin. The terminal sialic acid linkages differ among different glycoproteins. Thyroglobulin contains predominantly sialic acid α 2, 3 Gal and 2, 6 Gal in 2:1 ratio. The most common type of glycosidic linkage involving sialic acid in bovine submaxillary mucin (BSM) is α 2, 6 GalNAc. Although the terminal oligosaccharide sequence of BSM is NeuAC, α 2, 6 GalNAc, a major fraction (>50%) of the sialic acid was O-acetylated (Graham, 1966). Thus based on the above findings, sialic acid specificity of the agglutinin was tested and the results showed a reduction in HA titer with asialo buffalo erythrocytes confirming the agglutinin to be sialic acid specific. This must be due to the removal of sialic acids on buffalo erythrocytes which react with the agglutinin, by neuraminidase treatment (Esievo et al., 1982). Repeated adsorption with buffalo, dog, mice, rat and rabbit erythrocytes removed agglutinability of the crab serum suggesting the presence of a single hemagglutinin as reported in Episesarma tetragonum (Devi et al., 2013). Thus this study provides all the information necessary for the purification of the agglutinin using affinity chromatography. Purified agglutinin might provide precise information on its sugar specificity and biomedical applications.

REFERENCES

[1] Abhilash, J., and M Haridas, Metal ion coordination essential for specific molecular interactions of butea monosperma lectin: ITC and MD simulation studies, Applied Biochemistry and Biotechnology, Volume 176, Issue 1, pp 277-86, 2015. [2] Chien, J., S. Li, R. A. Laine and Y. Li, Characterization of gangliosides from bovine erythrocyte membranes, Journal of Biological Chemistry, Volume 253, Issue 21, pp 4031 – 4035, 1978. [3] Denis, M., and P.D. Mercy, Occurrence of agglutinin specific for sialogly coproteins in the hemolymph of the marine crab, Atergatis reticulates (De Haan), Journal of Ecobiology, Volume 11, Issue 2, pp 101-107, 1999. [4] Denis, M., S. Mullaivanam Ramasamy, B. Selvaputhiran Doss and K. Thayappan, Calcium dependent lectin in the serum of the marine crab Atergatis subdentatus (De Hann, 1835), Journal of Modern Biotechnology, Volume 5, Issue 5, 2016. [5] Devi, V., M. R. Basil Rose and P. D. Mercy, Sialic acid specific lectins from Episesarma tetragonum (Decapoda, Grapsidae): isolation, purification and characterization, International Journal of Aquatic Biology, Volume 1, Issue 4, pp 150-157, 2013. [6] Duk, M., and E. Lisowska, Effect of pH on the binding of Vicia graminea lectin to erythrocytes Dependence on the chemical character of red-cell receptors, European Journal of Biochemistry, Volume 143, pp 73-78, 1984.

JETIR1810299 Journal of Emerging Technologies and Innovative Research (JETIR) www.jetir.org 597

[7] Elayabharathi, T., and J. Vinoliya, Physico-chemical characterization of a hemolymph agglutinin from the marine crab, Atergatis ocyroe (Herbst, 1801). Crossian Resonance, Volume 8, Issue 1, pp 102-112, 2017. [8] Esievo, K.A.N., D.I. Saror, A. A. Ilemobade and M. H. Hallaway, Variation in erythrocyte surface and free serum sialic acid concentrations during experimental Trypanosoma vivax infection in cattle, Research in Veterinary Science, Volume 32, pp 1-5, 1982. [9] Gasmi, L., J. Ferre and S.Herrero, High bacterial agglutination activity in a single – CRD C-type lectin from Spodoptera exigua (Lepidoptera : Noctuidae), Biosensors, Volume 7, Issue 12, 2017. [10] Ghosh, J., C.M. Lun, A.J. Majeske, S. Sacchi, C.S. Schrankel and L.C Smith, Invertebrate immune diversity, Developmental and Comparative Immunology. Volume 35, pp 959-974, 2011. [11] Gowda, N.M., V. Goswami and M.I. Khan, Purification and characterization of a T-antigen specific lectin from the coelomic fluid of a marine invertebrate, seacucumber, Holothuria Scabra, Fish and Shellfish Immunology, Volume 24, pp 450-458, 2008a. [12] Graham, E.R.B., In: “Glycoproteins” (Gottschalk, A, ed) Elsevier publishing company, Amsterdam, pp 353-361, 1966. [13] Hamid, R., A. Masood, I.H. Wani and S.Rafiq, Lectins: Proteins with Diverse Applications, Journal of Applied Pharmaceutical Science, Volume 3 Issue 4, pp 93-103. 2013. [14] Hauzer, K., M. Ticha, V. Horejsi and J Kocourek, Studies on lectins. XLIV. The pH dependence of lectin interaction with sugars as determined by affinity electrophoresis, Biochimica et Biophysica Acta, Volume 583, Issue 1, pp 103 – 109, 1979. [15] Hosoi, T., Y. Imai and T. Irimura, Coordinated binding of sugar, calcium and antibody to macrophage C-type lectin, Glycobiology, Volume 8, Issue 8, pp 791–798, 1998. [16] Huet, M., Factors affecting the molecular structure and the agglutinating ability of concanavalin A and other lectins, European Journal of Biochemistry, Volume 59, Issue 2, pp 627-632, 1975. [17] Ishikawa, F., T. Kameyama, A. Takenaka, K. Oishi and K. Aida, Action of Proteases on Human Erythrocyte Glycoproteins in Relation to Hemagglutination by Conidiobolus Chitin-binding Agglutinin, Agricultural and Biological Chemistry, Volume 45, Issue 9, pp 2105-2110, 1981. [18] Kovar, V., P Kopacek and L.Grubhoffer, Isolation and characterization of Dorin M, a lectin from plasma of the soft tick Ornithodoros moubata. Insect Biochemistry and Molecular Biology, Volume 30, Issue 3 pp 195-205, 2000. [19] Krishnamoorthi, A., S. Selvakumar and G. Shanthi, Antimicrobial activity of lectins isolated from the haemolymph of marine crab Scylla serrata (Forskal 1775), International Journal of Modern Research and Reviews, Volume 4, Issue 11, pp 1350 – 1352, 2016. [20] Lis, H., and N.Sharon, Lectins as molecules and as tools, Annual Review of Biochemistry, Volume 55, pp 35-67, 1986. [21] Mercy, P.D., and M.H. Ravindranath, Purification and characterization of N-glycolyl neuraminic acid specific lectin from Scylla serrata, European Journal of Biochemistry, Volume 215, pp 697-704, 1993. [22] Mercy,P.D., and M.H. Ravindranath, An agglutinin with unique specificity for N-glycolyl sialic acid residues of thyroglobulin in the hemolymph of a marine crab, Scylla Serrata (Forskal), Experientia, Volume 48, pp 498-500, 1992. [23] Na, Y.J., Y.J. Kim, B.T. Park, B.W. Junk, K.W. Hwang and H. Kim, A novel lectin isolated from the hemolymph of the marine hair crab, Erimacrus isenbeckii. Protein and Peptide Letters, Volume 14, Issue 8, pp 800-803, 2007. [24] Neth, O., D.L. Jack, A.W. Dodds, H. Holzel , N.J. Klein and M.W. Turner, Mannose – binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition, Infection and Immunity, Volume 68, pp 688-693, 2000. [25] Pereira, M.E., A.F.B. Andrade and J.M.C. Ribeiro, Lectins of distinct specificity in Rhodnius prolixus interact selectively with Trypanosoma cruzi Science, Volume 211, pp 597 – 600, 1981. [26] Philip, A.O., P. Mullainadhan, S.M. Chrispinus and N.S. Donald, Characteristics of serum agglutinins in marine crab Scylla serrata Forskal and their interaction with various bacteria species, International Journal of Current Microbiology and Applied Science, Volume 2, Issue 10, pp 31-43, 2013. [27] Ravindranath, M.H., and J.C. Paulson, O-acetyl sialic acid specific lectin from the crab, Cancer antennarius, Methods in Enzymology, Volume 138 pp 520-527, 1987. [28] Reeves, R., and H.Rahn, Patterns in vertebrate acid - base regulation, In: Evolution of Repiratory Processes, a Comparative Approach (wood, S.C. and Lenfant, C., eds), Marcel Dekkar, New York, pp 225-252, 1979. [29] Salzet, M., Vertrbrate innate immunity resembles a mosaic of invetebrate immune responses, Trends in Iimmunology, Volume 22, Issue 6, pp 285-288, 2001. [30] Schauer, R., Chemistry, metabolism and biological functions of sialic acids, Advances in Carbohydrate Chemistry and Biochemistry, Volume 40, pp 131-234, 1982. [31] Scott, M.T., A naturally occurring agglutinin in the hemolymph of Periplaneta Americana, Archives de Zoologie Experimentale et Generale, Volume 112, pp 73-80, 1971. [32] Sharon, N., and H.Lis, Lectins as cell recognition molecules, Science, (1989). Volume 246, pp 227 – 234, 1989. [33] Sharon, N., Lectins: Past, present and future. Biochemical Society Transactions, Volume 36, pp 1457-1460, 2008. [34] Sheeja, V.U., Fresh water crab Travancoriana charu, (Bahir and Yeo, 2017) hemolymph lectin: Isolation, Purification and Possible Biological application. Ph.D Thesis Manonmaniam Sundaranar University, Tirunelveli, India. 2017. [35] Singh, A.P., and K.D. Saxena, Effect of temperature, pH and denaturing agents an biological activity of MCJ lectin, Chemical Science Transactions, Volume 2, Issue 4, pp 1508 - 1512. 2013. [36] Smith, V.J., Immunology of invertebrates: Cellular, In.els. John Wiley and Sons. Ltd, Chichestr, pp 1-13. 2016. [37] Sullivan, K , The lectin report. www.krispin.com/lectin.html. 2017. [38] Varki, A., and S. Kornfeld, (An autosomal dominant gene regulates the extent of 9-O-acetylation of murine erythrocyte sialic acids, Journal of Experimental Medicine, Volume 152, pp 532-544, 1980. [39] Yasue, S., S. Handa, S. Miyagawa, J. Inove, A. Hasegawa and T. Yamakawa, Difference in form of sialic acid in red blood cell glycolipids of different breeds of dogs, Journal of Biochemistry, Volume 83, pp 1101, 1978.

JETIR1810299 Journal of Emerging Technologies and Innovative Research (JETIR) www.jetir.org 598