© 2018 JETIR October 2018, Volume 5, Issue 10 www.jetir.org (ISSN-2349-5162) 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 © 2018 JETIR October 2018, Volume 5, Issue 10 www.jetir.org (ISSN-2349-5162) 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 © 2018 JETIR October 2018, Volume 5, Issue 10 www.jetir.org (ISSN-2349-5162) 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