J Arthropod-Borne Dis, June 2020, 14(2): 144–152 Z Nabavi et al.: Purification and Partial … 144 http://jad.tums.ac.ir Published Online: June 30, 2020 Original Article Purification and Partial Characterization of Agglutinin Lectin from Heamolymph of German Cockroach, Blattella germanica Zohreh Nabavi; Mozhgan Baniardalani; *Hamidreza Basseri Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (Received 23 Feb 2018; accepted 28 May 2020) Abstract Background: Lectin molecules have crucial biological role in insects’ immune system. The aim of present study was to find the agglutinin activities in haemolymph of German cockroach, Belatella germanica with appropriate screening and purification. Methods: The heamolymph of cockroach was collected and agglutinin test performed against different animal and human red blood cells (RBC). Then sugar inhibition assay was carried out to find carbohydrate specific binding lec- tin. The proteins of haemolymph was purified using ion-exchange chromatography (HPLC) and each fraction was tested for agglutinin activity. Finally the molecular weight of the agglutinin protein was determined using SDS-page. Results: The most agglutinin activity of haemolymph was found against RBC of mouse at titer 1/128ml/L dilution and sugar inhibition assay showed that fucos, N-acetyglucoseamine and galactose reduced titer of agglutinin to ½ml/L. Only one fraction of heamolymph at rotation time of 36 minute showed agglutinin activity. The molecular weight of this lectin was measured as 120Kds. Conclusion: The range of agglutinin activities against different RBC indicates that the isolated lectin is not specific for a particular carbohydrate. In addition, the isolated lectin at low concentration present in heamolymph should be an innate lactin not secreted, because we found it without any trigger immunity of the insect. Keywords: German cockroach; Heamolymph; Lectin Introduction Insects lack specific acquired nonself recog- nition systems such as antibody in vertebrates, and lectins molecules have main role of non- self recognition in the innate immune re- sponse (1-4). Lectins can agglutinate foreign matter and thus become to be it more suit- able for understanding. These molecules can also mediate haemocytes invasion to non- self particles, and consequently cause lysis, increase phagocytosis of foreign particles and activate the coagulation system (5, 6). Lectins are proteins or glycoproteins that recognize specifically and bind reversibly the carbohydrate-containing molecules of foreign cells (7-10). Lectin molecules have a range of immunologic action including antitumor, an- tifungi and bacteria activities which may find practical applications (11, 12). Carbohydrate- binding lectins with similar properties are pres- ence on the surfaces of many pathogens. There- fore, a wide variety of pathogens can be rec- ognized by specific binding of lectins (13, 14). However, the interaction between lec- tins and carbohydrates as ligand receptors is common in living organisms (15). It has been shown that two C-type lectins has roles in antibacterial defense as well as in the melanization response to Plasmodium berghei in Anopheles gambiae (16). In addi- tion, lectins as immune substances have been reported in American cockroach (Periplaneta americana) and desert locusts (Shistocerca gregaria) (17-19). Multiple lectins have been purified from only two species of cockroach- es, namely, the American cockroach, P. amer- icana; and the West Indian leaf cockroach, *Corresponding author: Dr Hamidreza Basseri, E-mail: Basserih@tums.ac.ir mailto:Basserih@tums.ac.ir J Arthropod-Borne Dis, June 2020, 14(2): 144–152 Z Nabavi et al.: Purification and Partial … 145 http://jad.tums.ac.ir Published Online: June 30, 2020 Blaberus discoidalis (20, 21). In addition, fucose-binding lectin in the heamolymph of P. americana showed the maximum phe- noloxidase activity against dopamine (6). Ac- cording to our knowledge and survey of lit- eratures, there are no many references exist about agglutinin activity in the heamolymph of German cockroach, Blattella germanica. Therefore, in the present study, we tried to identify and purify lectin molecule s from heamolymph of German cockroach. Our results provide a new insect lectin from B. germanica. It seems C-type lectin as recep- tors provide main role for penetration of cockroach allergens (22). This is the first report on characterization of hemagglutinin activity in German cockroach heamolymph. Hence, in the present study, we also attempt to isolate and purify lectin from the hea- molymph of B. germanica. The finding of current study provides initial information about presence of lectin the heamolymph of German cockroach. These results provide preliminary information for further studies such as allergenic actives cause by German cockroach. Materials and Methods Insect rearing and bleeding German cockroaches, Blatella germani- ca, were maintained in an insectary at 25±2 °C with humidity of 60–65%, and fed on dried bread, sugar cubic with water. Adult cockroaches (450 insects) were anaesthe- tized with CO2 and then the ventral surface of sternum of each cockroach was sterilized with 70% ethanol. The coxa membrane and ventral joint of abdomen to thorax were pierced with a sterile needle to pull out hae- molymph. The haemolyphm was collected, homogenized and then centrifuged at 1800 rpm for 15 minutes. The supernatant was kept in -80 °C until used. The protein con- centration of each sample was measured by Bradford assay method (23). Haemagglutination assay Haemagglutination assays were performed presented by Chen et al. (24). The red blood cells (RBCs) of rabbit, rat, sheep, Guinea pig, Syrian mouse and human (A, B, AB and O groups) were used to exam haemagglutina- tion activities of the haemolymph of B. ger- manica. All RBCs were washed three times in 10mM Phosphate Buffered Saline (PBS) containing 150mM NaCl and 10mM sodium phosphate at pH 7.4 and 380mOsm, 1mM CaCl2 at 4 ºC and the concentration of the RBCs suspensions to the buffer and adjusted to final concentration of 2%. Five microliters of the buffer was dis- pensed in each microtiter plate well and then two folded serial dilution of the haemolymph extract was prepared in each row of plates to give final dilution ranges of 2 -1 to 2 -10 , prior to the addition of 5μl RBCs to each well. The plates were covered and kept at room temperature for two hours. The end points of agglutination examined under a stereomicro- scope and by the flow characteristics of the erythrocyte pellets when the plate was held at an angle. All experiments were replicated three times and the controls always included PBS plus RBCs alone. Carbohydrate inhibition assays Carbohydrate inhibition assays were fol- lowed as mentioned by Chen et al. (24). The sugar specificities of cockroach lectins were investigated by competitive binding using the following carbohydrates: D-(+)-glucose, D-(+)-galactose, D-(+)-mannose, L-(-)-fucose, lactose, N-acetyl-D-glucosamine, N-acetyl- D-galactosamine, fructose and arabinose (all purchased from Sigma Co.). Stock solutions of sugars were prepared in PBS at 0.2M and stored in -4 °C until use. Two folded serial dilutions of haemolymph (each of 5μl) were prepared in PBS followed by addition of 5μl of appropriate carbohydrate at the above ini- tial concentration. The plates were incubated at room temperature for 60min and then 5μl J Arthropod-Borne Dis, June 2020, 14(2): 144–152 Z Nabavi et al.: Purification and Partial … 146 http://jad.tums.ac.ir Published Online: June 30, 2020 of the mouse RBCs added to the respective wells. The controls were consisted of carbo- hydrate plus PBS, and plus RBCs alone. In- hibitory effects were recorded as those re- ducing agglutination in the wells. The end points of agglutination were examined under a stereomicroscope and by the flow charac- teristics of the erythrocyte pellets when the plate was held at an angle. This experiment was replicated three times. Lectin purification Ion exchange-High Performance Liquid Chromatography (HPLC) (Knauer Co., Ger- many) was utilized to separate protein from extracted haemolymph. The samples once more were centrifuged for 15 minutes at 1800 rpm and supernatant filtered three times through a 0.45µm filter. The sample was load- ed to column resin (Asahi chemical Co., Japan) with carboxymethyl as the functional group as particles, 7.5×100 ID, 150mm length, 2000 Pore size). The mobile phase was 1M of NaCl in 20mM Tris-HCl (pH= 7.5–8) which applied at a flow rate of 1ml/min at room temperature. The column was previously equil- ibrated with 20mM Tris-HCl buffer at vol- ume 300µL from 0–20min and a flow rate of 1.0ml/min. The sample at 300µL volume was loaded to an ion- exchange column. The pro- teins were eluted with a 50min linear gra- dient of 90–10% NaCl/Tris-HCl at a flow rate of 1.0ml/min at room temperature. The chromatographic run was monitored at 280 nm of absorbance. The fractions were collected manually and then the eluted fractions were concentrated using freeze-dryer device. Finally agglutinin activity of each eluted protein was assayed. The molecular weight of agglutinin protein was measured by polyacrylamide gel elec- trophoresis (SDS-PAGE). Polyacrylamide gel electrophoresis (SDS- PAGE) The molecular weight of protein, purity and molecular mass of fractions were esti- mated using SDS-page. Tris base 30.3g, Gly- cine 144g, SDS 10g and make to 1L with dH2O. Twenty μL of the eluted protein were loaded on the SDS-PAGE gels (12%) and run. The gel was stained with Coomassie brilliant blue R 250/Silver. As well as stand- ard protein marker was loaded on SDS- PAGE (mix of seven proteins, 14.4–116KDa). BioRad apparatus was used, the gels were run at 200V (constant voltage) until the bro- mophenol blue dye was just off. Gels were run at room temperature and 50–60 minutes. Ethics approval Ethics approval was not required for this study. Results Haemagglutination assay The results of haemagglutination activities of whole cockroache serum against a range of erythrocytes were represented in Fig. 1. The highest activity were found against mouse erythrocytes (titer ≤128) followed by Sheep (titer ≤ 64) but these activities were relative- ly less against different human erythrocytes (Fig. 1). The lowest activity was observed with human O- erythrocytes (titer ≤8). Thus, Mouse erythrocytes were candidate for fur- ther inhibition assays. Carbohydrate inhibition assay The results of haemagglutination inhibi- tion assay are represented in Fig. 2. The hae- molymph lectin activity was reduced by all tested sugars except fructose. The inhibitory effect of Galactose, fucose and N-acetyl-D- glucosamine was more than other carbohy- drates while the agglutination of hemolymph reduce to titer of ≤2. Fructose did not show any specific binding to lectins of haemo- lymph. Glucose, mannose, lactose and N-ac- etyl-D-galactosamine showed similar inhibi- tory effect on the lectin activity at titer of ≤8 J Arthropod-Borne Dis, June 2020, 14(2): 144–152 Z Nabavi et al.: Purification and Partial … 147 http://jad.tums.ac.ir Published Online: June 30, 2020 which indicate that non-specific binding ex- ist in the haemolymph lectins for these car- bohydrates. Arabinose also blocked aggluti- nin activity at titer of ≤ 4 which it was mod- erate inhibitor. Ion-exchange Chromatography Following the separation and purification of the protein molecules using ion exchange HPLC, five peaks at chromatogram were ap- peared. All fractions were applied for hae- magglutination activities and the main activ- ity was found by fifth peak which appeared at 36min retention time (tR) in chromatogram (Fig. 3). The molecular weight of the puri- fied protein was estimated to be about 66 kDa by sodium dodecylsulfate polyacryla- mide gel electrophoresis (Fig. 4). Fig. 1. Agglutination activities of haemolymph of the German cockroach, Blattela germanica, against different red blood cell (RBC) at 2% concentration in phosphate buffered saline. All experiments were replicated three times and the control was Phosphate Buffered Saline (PBS) free haemolymph J Arthropod-Borne Dis, June 2020, 14(2): 144–152 Z Nabavi et al.: Purification and Partial … 148 http://jad.tums.ac.ir Published Online: June 30, 2020 1 2 4 8 16 H A a ct iv it y ( Lo g 2 ) Fig. 2. Haemagglutination inhibition assay of hemolymph lectins of the German cockroach, Blattela germanica. The assay was performed with mice Red Blood Cells (RBCs) (2%) in Phosphate Buffered Saline (PBS). The control was haemolymph free carbohydrate Fig. 3. Ion exchange- High Performance Liquid Chromatography (IE-HPLC) of Blattela germanica hemolymph. 300µL of the diluted hemolymph was loaded into the column (7.5×100 ID, 150mm length, 2000 A° Pore size). The column was equilibrated with 300µL water from 0–20min at a flow rate of 1.0ml/min. Proteins were eluted with a 50min linear gradient of 90–10% NaCl / Tris-HCl at a flow rate of 1.0ml/min at room temperature. The chromato- graphic run was monitored at 280nm of absorbance. Five peaks were observed in chromatogram, Only fifth peak was determined presence of lectin in hemagglutination assay J Arthropod-Borne Dis, June 2020, 14(2): 144–152 Z Nabavi et al.: Purification and Partial … 149 http://jad.tums.ac.ir Published Online: June 30, 2020 Fig. 4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole heamolymph proteins of Belattella germanica and the fractions which showed agglutinin activity. All fractions prepared using Reversed phase High Performance Liquid Chromatography (RP-HPLC) and applied for agglutinin activity assay. The molecu- lar weight of isolated lectin was approximately 66KDa. Gel was stained with Coomassie brilliant blue R 250 Discussion In the current study, we assessed haemag- glutinin activity in the heamolymph of Ger- man cockroach. The mouse and sheep eryth- rocytes showed high agglutinin reaction with the heamolymph lectins. Similarity, the eryth- rocytes of mice showed high agglutinin ac- tivity against heamolymph of discoid cock- roach, Blaberus discoidalis. Therefore, the au- thors used the mice erythrocytes for study on two isolated lectins called as BDL1 and BDL2 (24). In the current study, we also used mouse erythrocytes for inhibition assays. German cockroaches have developed ef- fective innate immunity of protecting them- selves against pathogenic microorganisms in- cluding epidermal immunity (25) or humoral immune defense (24). Lectins are one of the pattern-recognition proteins concerned in innate immunity in in- sects (4). The presence of lectin activities in the heamolymph is more important for immun- ity of B. germanica against microbial patho- gens. As recently showed, C-type lectin has crucial role to response to pathogen infec- tion by the expression of antimicrobial pep- tides and the agglutination of bacteria im- munity in red flour beetle Tribolium casta- neum (3). Therefore, further work needs to characterize the role of lectins in immunity of the German cockroach. We optimized ion exchange-HPLC to pu- rify that protein which showed agglutinin activities. The multiple lectins had been pu- rified from different species of cockroaches e.g. the American cockroach, Periplaneta americana (6, 19, 25-27) and the West Indi- an leaf cockroach, Bl. discoidalis (21). At least four lectins have been reported in this species, namely, BDL1, BDL2, BDL3, and GSL (24). In addition, based on carbohydrate specificity, we characterized specificity of pro- tein binding carbohydrates to the hemolymph 66 KDa J Arthropod-Borne Dis, June 2020, 14(2): 144–152 Z Nabavi et al.: Purification and Partial … 150 lectins of the German cockroaches using dif- ferent carbohydrates. The lectins of German cockroach showed more affinity to glucose, N-acetyl-D-gluctosamine and fucose. Based on Makela category (28), in the current study, the lectin in B. germanica heamolymph could be categorized in groups II and III. These types of lectins are more involve in immun- ity of insects and have crucial role in phago- cytosis of microorganisms (20). In addition, the heamolymph lectin of B. germanica agglutinated different types of an- imal and human blood cells which indicated this lectin is non-blood type–specific. Gen- erally, lectins recognized α-linked on carbo- hydrate surface of red blood cells, as we could not find RBC specificity, it may indi- cate the lectins failed to distinguish between α and ß-linked of carbohydrates on the sur- face of the blood cells. It was also possible that the lectin have independent binding sites, which can be used for attachment to the erythrocyte surface. In the present study, only one fraction of the heamolymph with molecular weight of 66KDa (with no subunits) showed agglutinin activities against mice RBC (Fig. 1). Gener- ally, the molecular weights of all lectins of invertebrates vary from 26KDa to 1500KDa (29-31). This range of difference could be depended on species or methods used for pu- rification, analysis and protocols. The max- imum molecular weight of insect lectin (1500 KDa) was reported in P. americana (25) whereas the lowest molecular weight lectin was that of Agrotis segetum with 69KDa and no subunits (32) while we introduce a lectin from heamolymph of B. germanica with mo- lecular weight of 66KDa. According to our results, this agglutinin activities could be cal- cium dependant-lectin. While the C-type lec- tin family consists of members that bind their ligands in a calcium-dependent manner, many other C-type lectins show the hemoagglutinin activity in a calcium-independent manner. Conclusively, our current results provide a new insect lectin from German cockroach, B. germanica. This finding would be helpful in future studies on lectins concerning. Acknowledgements The authors would like to thank Dr Behrouz Vaziri and Mrs Fatemeh Torkashvand in pro- tein chemistry lab, Pasteur Institute of Iran for their valuable helps. This study was sup- ported by the Deputy of Research, Tehran University of Medical Sciences, Grant No. 19502 References 1. Arumugam G, Sreeramulu B, Paulchamy R, Thangavel S, Sundaram J (2017) Purification and functional character- ization of lectin with phenoloxidase activity from the hemolymph of cock- roach, Periplaneta americana. Arch Insect Biochem Physiol. 95(2): e21390. 2. Basseri HR, Doosti S, Akbarzadeh K, Nateghpour M, Whitten MM, Ladoni H (2008) Competency of Anopheles stephensi mysorensis strain for Plas- modium vivax and the role of inhibi- tory carbohydrates to block its sporo- gonic cycle. Mal J. 7: 131. 3. Basseri HR, Emmami, N, Haji-Hosseini R, Abolhasani M, Moradi A (2008) Biological transmission of bacteria in- hibit by hemolymph lectins of Amer- ican cockroach. Iranian J Public Health. 37: 75–82. 4. Basseri HR, Mohamadzadeh Hajipirloo H, Mohammadi Bavani M, Whitten MM (2013) Comparative susceptibility of different biological forms of Anophe- les stephensi to Plasmodium berghei anka strain. Plos One. 8: E75413. 5. Bi J, Feng F, Li J, Mao J, Ning M, Song X, Xie J, Tang J, Li B (2019) A c- type lectin with a single carbohydrate- J Arthropod-Borne Dis, June 2020, 14(2): 144–152 Z Nabavi et al.: Purification and Partial … 151 recognition domain involved in the innate immune response of Tribolium castaneum. Insect Mol Biol. 28(5): 649–661. 6. Bradford MM (1976) A rapid and sensi- tive method for the quantitation of microgram quantities of protein uti- lizing the principle of protein-dye bind- ing. Anal Biochem. 72: 248–254. 7. Bulgakov AA, Park KI, Choi KS, Lim HK, Cho M (2004) Purification and char- acterization of a lectin isolated from the manila clam Ruditapes philippinar- um in Korea. Fish Shellfish Immu- nol. 16: 487–499. 8. Chen C, Durrant HJ, Newton RP, Ratcliffe NA (1995) A study of novel lectins and their involvement in the activa- tion of the prophenoloxidase system in Blaberus discoidalis. Biochem J. 310(1): 23–31. 9. Chen C, Ratcliffe NA, Rowley AF (1993) Detection, isolation and characteriza- tion of multiple lectins from the haemo- lymph of the cockroach Blaberus dis- coidalis. Biochem J. 294(1): 181–190. 10. Chen C, Rowley AF, Newton RP, Ratcliffe NA (1999) Identification, purification and properties of a beta-1,3-glucan- specific lectin from the serum of the cockroach, Blaberus discoidalis which is implicated in immune defence re- actions. Comp Biochem Physiol B Bi- ochem Mol Biol. 122: 309–319. 11. Do DC, Zhao Y, Gao P (2016) Cockroach allergen exposure and risk of asthma. Allergy. 71: 463–474. 12. Drickamer K, Taylor ME (1993) Biolo- gy of animal lectins. Annu Rev Cell Biol. 9: 237–264. 13. Gül N, Ayvali C (2002) Purification and determination of the molecular struc- ture of hemolymph lectin of Agrotis segetum (Denis and Schiff). Turk J Biol. 26: 49–55. 14. Kawasaki K, Kubo T, Natori S (1993) A novel role of Periplaneta lectin as an opsonin to recognize 2-keto-3-deoxy octonate residues of bacterial lipopol- ysaccharides. Comp Biochem Phys- iol B. 106: 675–680. 15. Kawasaki K, Kubo T, Natori S (1996) Presence of the Periplaneta lectin-re- lated protein family in the american cockroach Periplaneta americana. In- sect Biochem Mol Biol. 26: 355–364. 16. Kubo T, Kawasaki K, Natori S (1993) Transient appearance and localiza- tion of a 26-kda lectin, a novel mem- ber of the Periplaneta lectin family, in regenerating cockroach leg. Dev Biol. 156: 381–390. 17. Kubo T, Natori S (1987) Purification and some properties of a lectin from the hemolymph of Periplaneta americana (american cockroach). Eur J Biochem. 168: 75–82. 18. Lackie AM, Vasta GR (1988) The role of galactosyl-binding lectin in the cel- lular immune response of the cock- roach Periplaneta americana (Dic- tyoptera). Immunology. 64: 353–357. 19. Lam SK, Ng TB (2011) Lectins: produc- tion and practical applications. Appl Microbiol Biot. 89: 45–55. 20. Makela O (1957) Studies in hemaggluti- nins of leguminosae seeds. Ann Med Exp Biol Fenn. 35: 1–133. 21. Olafsen JA (1995) Lectins: models of natural and induced molecules in in- vertebrates. In: Cooper EL (Ed) Ad- vances in Comparative Environmen- tal Physiology. Springer-Verlag, Ber- lin, pp. 49–76. 22. Ottaviani E, Malagoli D, Franchini A (2004) Invertebrate humoral factors: cytokines as mediators of cell sur- vival. Prog Mol Subcell Biol. 34: 1– 25. 23. Ratcliffe A, Fryer PR, Hardingham TE (1985) Proteoglycan biosynthesis in chondrocytes: protein a-gold locali- J Arthropod-Borne Dis, June 2020, 14(2): 144–152 Z Nabavi et al.: Purification and Partial … 152 zation of proteoglycan protein core and chondroitin sulfate within golgi sub compartments. J Cell Biol. 101: 2355–2365. 24. Schnitger AK, Yassine H, Kafatos FC, Osta MA (2009) Two c-type lectins cooperate to defend Anopheles gam- biae against gram-negative bacteria. J Biol Chem. 284: 17616–17624. 25. Thakur K, Kaur T, Kaur M, Hora R, Singh J (2019) Exploration of carbo- hydrate binding behavior and anti-pro- liferative activities of Arisaema tortu- osum lectin. BMC Mol Biol. 20: 15. 26. Viswambari Devi R, Basilrose MR, Mer- cy PD (2010) Prospect for lectis in arthropods. Ital J Zool. 3: 257–260. 27. Ward SE, O'Sullivan JM, Drakeford C, Aguila S, Jondle CN, Sharma J, Fal- lon PG, Brophy TM, Preston RJS, Smyth P, Sheils O, Chion A, O'Don- nell JS (2018) A novel role for the macrophage galactose-type lectin re- ceptor in mediating von willebrand factor clearance. Blood. 131: 911–916. 28. Wilson R, Chen C, Ratcliffe NA (1999) Innate immunity in insects: the role of multiple, endogenous serum lectins in the recognition of foreign invaders in the cockroach, Blaberus discoidalis. J Immunol. 162: 1590–1596. 29. Xia X, You M, Rao XJ, Yu XQ (2018) Insect c-type lectins in innate immun- ity. Dev Comp Immunol. 83: 70–79. 30. Yu XQ, Kanost MR (2000) Immulectin- 2, a lipopolysaccharide-specific lec- tin from an insect, Manduca sexta, is induced in response to gram-negative bacteria. J Biol Chem. 275: 37373– 37381. 31. Yu XQ, Kanost MR (2003) Manduca sexta lipopolysaccharide-specific immulec- tin-2 protects larvae from bacterial in- fection. Dev Comp Immunol. 27: 189– 196. 32. Zheng X, Xia Y (2012) Beta-1, 3-glucan recognition protein (betagrp) is essen- tial for resistance against fungal path- ogen and opportunistic pathogenic gut bacteria in Locusta migratoria ma- nilensis. Dev Comp Immunol. 36: 602– 609.