Characterization and Purification of an Agglutinin from the Hemolymph of the Crab Oziotelphusa Cf. Hippocastanum
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© 2018 IJRAR December 2018, Volume 5, Issue 4 www.ijrar.org (E-ISSN 2348-1269, P- ISSN 2349-5138) Characterization and purification of an agglutinin from the hemolymph of the crab Oziotelphusa cf. hippocastanum 1 Sheeja V U, 2 Basil Rose M R 1 Research Scholar, 2 Associate Professor 1 Department of Zoology, Holy Cross College, Nagercoil, Tamilnadu, India Abstract: A hemagglutinin isolated and purified by adsorption on formalinized erythrocytes from the hemolymph of the freshwater crab Oziotelphusa cf. hippocastanum showed high specificity for buffalo and rabbit erythrocytes among the erythrocytes tested. The hemolymph agglutinin was calcium dependent and was highly sensitive to the changes in pH and temperature of the medium. Cross adsorption assay revealed the presence of a single agglutinin which was strongly inhibited by sialic acid specific glycoproteins, bovine thyroglobulin > fetuin and sugars D-galactosamine > N-acetyl–D-Glucosamine and N-acetyl-D- Mannosamine .The molecular weight of the lectin was 68 kDa. IntexTerms: Crustacea, lectin, hemagglutinin, sialoglycoprotein I. 1NTRODUCTION Crustaceans lack adaptive immune system and rely exclusively on their innate immune mechanisms that include both cellular and humoral responses [1]. Lectins act as humoral factors in the defense mechanism, the same way as immunoglobulins in vertebrates. Lectins are proteins or glycoproteins normally without catalytic activity that can recognize and non-covalently bind to specific sugar moieties and thereby agglutinate cells by binding to cell surface glycoproteins and glycoconjugates [2]. Because of their specificity for certain sugar moieties, lectins are usually defined in terms of their ability to agglutinate erythrocytes in hemagglutination tests. Those reacting with the erythrocytes do so by virtue of the presence of carbohydrate configurations or antigenic determinants of the human blood group substances [3]. Many kinds of microorganisms, including bacteria share surface structure components with vertebrate erythrocytes. Lectins, therefore, are considered important pattern recognition proteins in innate immunity and play significant role in non-self recognition and clearance of invading microorganisms, either as cell surface receptors or as soluble proteins existing in circulating fluid [4, 5]. A large number of natural lectins have been purified and characterized by biochemical methods from hemolymph of crustaceans. Isolation of lectins is usually carried out by affinity adsorption either on a chromatographic matrix (i.e.sepharose) derivatized with glycoconjugates or on red blood cells (RBC) fixed chemically [6, 7]. Reitherman reported purification using formalinized erythrocytes as a general affinity adsorbent. This present study describes the physio–chemical properties of the hemolymph agglutinin/lectin of the freshwater crab Oziotelphusa cf. hippocastanum and its isolation and purification using formalinized buffalo erythrocytes. II. MATERIALS AND METHODS Collection of hemolymph Hemolymph was collected from the cut end of the dactylus region of the leg in sterile plastic tubes held on ice and used immediately. Preparation of erythrocytes Human and blood samples of various animal species were collected by venous or cardiac puncture in cold Alsevier’s medium containing 30 mM sodium citrate pH 6.1, 77 mM, NaCl, 114 mM dextrose, 100 µg/ml of neomycin sulphate and 330 µg/ml of chloremphenicol. Subsequently the erythrocytes were washed twice with 0.9% saline and once with TBS (50 mM Tris, 100 mM NaCl, and 10 mM CaCl2) and formulated in the same to get 1.5% erythrocyte suspension. Hemagglutination assay (HA) Hemagglutination assay was performed in U-bottom microtiter plates by serial dilution of 25 µl of sample with an equal volume of TBS (Tris Buffered Saline 50 mM Tris 100 mM NaCl, 10 mM CaCl2) followed by the addition of 1.5% buffalo erythrocyte suspension. Positive agglutination was obtained when the erythrocytes did not sediment to the bottom of the well forming a red button. HA titer was recorded as the highest dilution that still caused agglutination. To determine the physio-chemical parameters, HA assay was performed at various pH of the TBS and varying temperatures (0 -100ºC). To test the effect of various concentration of cations and chelators (disodium, tetrasodium EDTA and trisodium citrate), the hemolymph was serially diluted with TBS containing various concentrations of Ca2+ or Mg2+ or Mn2+ and EDTA (0.01,0.1, 1, 5, 10, 20, 30, 40, 50, 100 mM). HA was determined using 1.5% buffalo erythrocyte suspension after incubation at room temperature for one hour. Cross adsorption assay Buffalo, rabbit, rat, pig, mice and dog erythrocytes were washed and packed. (Twice saline wash followed by TBS wash). Hemolymph was mixed with equal volume of packed erythrocytes and incubated for 18 hours at 4ºC with occasional shaking. The sample was centrifuged at 4000 x g for 5 minutes and the supernatant was collected for hemagglutination assay. Hemagglutination inhibition assay (HAI) Sugar specificity of the hemolymph agglutinin was determined by competitive inhibition using sugars and glycoproteins. For inhibition assay 25 µl of various sugars or glycoproteins were serially diluted with TBS. To this 25 µl of hemolymph diluted to subagglutination IJRAR1944549 International Journal of Research and Analytical Reviews (IJRAR) www.ijrar.org 226 © 2018 IJRAR December 2018, Volume 5, Issue 4 www.ijrar.org (E-ISSN 2348-1269, P- ISSN 2349-5138) concentration (hemolymph diluted with TBS which shows a HA of 2 wells) was added. The minimum concentration of the inhibitor required to completely block agglutination was observed after one hour of incubation at room temperature using 1.5% buffalo erythrocyte suspension. Purification using formalinized erythrocytes as the affinity adsorbent Formalinized buffalo erythrocytes were prepared following the procedure of Nowak and Barondes [8]. Erythrocytes were washed three times in 20 volumes of PBS, pH 7.2 (75 mM NaCl, 75 mM Na2HPO4) per packed cell volume by centrifugation at 1000 x g for 5 minutes. The cells were suspended at a concentration by volume of 8% PBS (pH 7.2) and an equal volume of formalin (3% solution in PBS with pH adjusted to 7.2 and 0.1 M NaOH) was added. The mixture was incubated at 37ºC for 16 hours with moderate shaking. The cells were then washed four times in five volumes of PBS (pH 7.2) per packed cell volume and then washed six times in 10 volumes of TBS, pH 7.2 (50 mM Tris HCl, 100 mM NaCl). The packed cells were incubated with 20 volumes of the clarified hemolymph in plastic tube for 2 hours with moderate shaking at 4ºC and again washed 3 times with 20 volumes of TBS, pH 7.2, containing 0.01M CaCl2. The cells were incubated with 10 volumes of 10 mM disodium EDTA in TBS, pH 8.2. After 10 minutes the elution mixture was certifuged at 28000 x g to remove any residual particulate material 2+ and the resultant supernatant was dialyzed against 10 mM CaCl2 (Tris/Ca ). Polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed on 12% slab gels according to the principle of Laemmli [9]. Sample was solubilized by boiling for 2 minutes in 1M Tris HCl buffer (pH 6.8, 10% SDS, 2% β mercaptoethanol, 15% glycerol and bromophenol blue). Each well was loaded with 25 µl of the sample and electrophoretical separations were performed in Tris tank buffer (Tris, Glycerine, SDS). A standard molecular weight protein marker was used as standard. Gels were stained with Coomassie brilliant blue (R-250). III. RESULTS HA assay The hemolymph of Oziotelphusa cf. hippocastanum showed diverse binding specificities with the different erythrocytes assayed. The agglutination titer was buffalo > rabbit erythrocytes followed by mice > dog. The HA titer for rat, pig, guinea pig and horse was comparatively less. Table 1. Hemagglutination titer of the hemolymph of the crab Oziotelphusa cf. hippocastanum against mammalian erythrocytes Erythrocytes n=5 HA titer Buffalo 512 Rabbit 256 Mice 256 Dog 128 Rat 32 Pig 32 Guinea pig 32 Horse 32 Human A 8 Human B 8 Human O 8 Cow 8 Goat 2 n= number of crabs tested pH and thermal stability Hemolymph agglutinin had maximum activity at the optimum pH 7.5. Highest hemagglutination titer was observed at the optimum temperature of 30ºC-40ºC but the activity decreased markedly between 50ºC and 70ºC, the activity was completely lost beyond 70ºC. Table 2. Effect of pH and temperature on hemagglutination titer of the freshwater crab Oziotelphusa cf. hippocastanum pH (n =10) HA titer Temperature (n =10) HA titer 5.0 128 0 256 5.5 128 10 256 6.0 256 20 256 6.5 256 30 512 7.0 256 40 512 7.5 512 50 256 8.0 256 60 128 8.5 128 70 4 9.0 128 80 2 9.5 64 90 0 10.0 64 100 0 n= number of crabs tested IJRAR1944549 International Journal of Research and Analytical Reviews (IJRAR) www.ijrar.org 227 © 2018 IJRAR December 2018, Volume 5, Issue 4 www.ijrar.org (E-ISSN 2348-1269, P- ISSN 2349-5138) Effect of divalent cations and chelators Highest hemagglutination titer was observed at 1-5 mM Ca2+ and 1 mM concentration of EDTA di- and tetra sodium salts. Table 3. Effect of cations (Ca2+, Mg2+ and Mn2+) on the hemagglutinating activity of the hemolymph agglutinin of the crab Oziotelphusa cf. hippocastanum Concentration of HA titer cations (mM) n =5 Ca2+ Mg2+ Mn2+ 0 128 128 128 0.01 256 128 256 0.1 256 128 256 1 512 128 256 5 512 128 128 10 128 128 128 20 64 128 128 30 32 64 128 40 32 32 128 50 32 32 64 100 32 32 32 n = number of crabs tested Table 4. Effect of chelators on the HA titer of hemolymph agglutinin of Oziotelphusa cf. hippocastanum Concentration HA titer of EDTA (mM) Disosodium Tetrasodium Trisodium n =5 salt salt citrate 0 128 128 128 0.01 128 128 128 0.1 128 256 128 1 512 512 128 5 256 128 128 10 4 4 128 20 4 4 128 30 4 2 256 40 2 2 128 50 2 2 128 100 2 2 64 n = number of crabs tested Cross adsorption assay Cross adsorption assay revealed the presence of a single agglutinin because it failed to agglutinate the erythrocytes after the second round of adsorption.