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Serological characterization of humoral lectin from Heterometrus granulomanus scorpion hemolymph
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. i0, pp. 295-304, 0145-305X86 $3.00 + Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd, All rights reserved.
SEROLOGICAL CHARACTERIZATION HETEROMETRUS GRANULOMANUS
1986.
OF HUMORAL LECTIN FROM SCORPION HEMOLYMPH*
H. AHMED 1 G. ANJANEYULU 2 and B P.CHATTERJEE 1 iDepartment of Macromolecules, Indian Association for the Cultivation of Science, Jadavpur, Calcutta - 700 032 India 2Department of Zoology, Hindu College, Guntur - 522 002,India
ABSTRACT
A lectin in the hemolymph of Indian scorpion Heterometrus sran ulomanus was detected by agglutination of human and animal erythrocytes. The agglutinating activity was enhanced in presence of Ca2+ ion. The lectin shows specificity for sialic acid like many other sialic acid-specific lectins such as "Limulin", as the agglutination of erythrocytes was completely abolished by treatment with Vibrio cholerae neuraminidase. However, neuraminidase-treated rat and mouse erythrocytes exhibited high titers. This result was substantiated by crossed-absorption test which suggests the adjunct specificity of the Heterometrus lectin for these cells. Hemagglutination-inhibition results of the lectin indicate that sialic acid including its derivatives and sialoglycoconjugates are the inhibitors among which glycophorin was most potent.
INTRODUCTION
Noguchi first observed the presence of hemagglutinin in the coelomic fluid of the horseshoe crab Limulus polyphemus(1). Similar substances, generally now called lectins (2) have gradually been discovered in the hemolymphs and tissue extracts of many invertebrate species, as well as in plants (3-5) and vertebrates (6). Lectins define a class of heat sensitive proteins or glycoproteins which, because of their polyvalent configuration and binding affinity for specific cell surface carbohydrates, can selec-
* Part of this work was presented at the XIIth International Symposium held in Utrecht, July, 1984. 295
Carbohydrate
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tively agglutinate vertebrate erythrocytes. This agghJtination is usually dependent upon the presence of divalent metal ions which apparently stabilize tertiary conformation of lectin molecules (7). They occur as fundamental biological principle in all living systems like in viruses, bacteria and parasites, plants, invertebrates and vertebrates and play an essential role, which may be different in all these systems according to diverse functions. For instance in bacteria they are helpful generally for causing an infection by mediating adhesion to the cell surface through a specific carbohydrate receptor (8). Functional knowledge of invertebrate lectins is rudimentary and many suggestions on them are offered though they are not experimentally verified. Some of these are (i) receptor recognition that is recognition between self and nonself (ii) recognition and removal of aged glycoconjugates and (iii) defence against foreign invaders. Though the study of serum lectins from the chelicerates was pioneered by Noguchi at the begining of this century, complete characterization of Limulus polyphemus hemagglutinin and its specificity for sialic acids was demonstrated by Cohen (9), P~rdoe et al. (i0) and Roche and Monsigny (11). Other Chelicerata studied are Japanese horseshoe crab Tachypleus tridentatus (12,13) and the Indian horseshoe crab Carcinoscorpius rotunda cauda (14). The former exhibits two groups of lectins specific for several N-acylamino sugars and sialic acids whi]e the latter binds sialic acid only. Anti-sialyl lectins have also been found in the Crustacea - the lobster Homarus americanus (15,16) and the coconut crab Birgus latro (17). Within the class Scorpiones, agglutinins were detected in the hemolymph of Saharan scorpion Androctonus australis (18,19) and the Arizona lethal scorpion Centruroides sculpturatus (20), whip scorpion Mastigoproctus giganteus (21).
In the present communication we report different agglutination profiles of Heterometrus hemolymph and its specificity.
MATERIALS AND METHODS
Agglutinins Heterometrus granulomanus scorpions were procured from Andhra Pradesh, India and identified by Zoological Survey of India, Calcutta. The hemolymph from both sexes of living specimens was collected from pedipalp by using 20-gauge needle. The hemolymph was allowed to clot at 25 °C for 1 hour and then centrifuged at 12,000 x g for 30 minutes in the cold. The clear supernatant was preserved at -20 °C until use.
Inhibitors N-acetylneuraminic acid (NANA), N-glycolylneuraminic acid (NGNA), NANA-lactose, N-acetyl-D-glucosamine (GIcNAc), N-acetyl-D-galactosamine (GalNAc), Digalactose, peptone A, IgD/Fc, fetuin, colominic acid, human glycoprotein fraction VI were purchased from Sigma Chemical Co. U.S.A. Ovarian A and H substances, bird nest glycoprotein and pig erythrocyte mucoid were kindly donated by Prof. G. Uhlenbruck, Medical University
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Clinic, Cologne, F.R.G. Glycophorin was the kind gift of Dr. A.K. Shukla, Biochemisches Institut, Christian-Albrechts-Universitat, Kiel, F.R.G. Diand mono-O-acetyl-N-acetylneuraminic acid, fluoro-N-acetylneuraminic acid were obtained from Dr. T.K. Ghosh, The Johns Hopkins University, Department of Biology, Baltimore, U.S.A. All other reagents were from Merck or B.D.H.
Erythrocytes Human blood was obtained from healthy donors by vein puncture and was collected in citrate-dextrose solution. Rat, mouse and rabbit blood was obtained either by vein or cardiac puncture. The blood of duck and pigeon was obtained by sacrificing the animals and that of the rest animals was collected from slaughter house. The erythrocytes were washed thrice with saline 0.85% (w/v) and twice with TBS (Tris-buffered saline; I00 mM TrisHCI, 150 mM NaCI, pH 7.6) and suspended at concentrations of 5 x 106 RBCs/ ml in TBS.
Enzymes Pronase P from Streptomyces griseus was purchased from Serva, Heidelberg, F.R.G. Neuramini'dase from Vibrio cholerae (VCN) 500 u/ml was from Behring Werke AG, Marburg, F.R.G. The RBCs were treated with pronase P and VCN according to Chatterjee et al. (22).
Hemagglutination Test This was performed in Takatsy microtiter plates. To two-fold serially diluted Heterometrus hemolymph (25 ~i) in TBS was added equal volumes of the erythrocyte suspension. The plates were incubated at 27 °C for 30 minutes after gentle shaking. Agglutination was read visually. The reciprocal of the highest dilution of the hemolymph showing visible agglutination was recorded as the titer. The experiments were performed in duplicate and controls were set up using TBS instead of hemolymph.
Crossed-absorption Test Diluted hemolymph was mixed with one-third of its volume in washed, packed and enzyme-treated RBCs and incubated for 45 minutes with occssional shaking. After centrifugation at 1000 x g for 5 minutes the supernatant was titrated as described above.
Hemagglutination-inhibition Test All inhibit0rs to be tested were dissolved in TBS at concentrations up to 200 mM for mono- and oligosaccharides and i% (w/v) for po]ysaccharides and glycoproteins adjusted at pH 7.6 with concentrated NaOH and ser~a-fly two-fold diluted in TBS in microtiter p]ates~ Eq,al volumes of diluted hemolymph (Titer 2) were added and incubated for ] hour at room temperature. The mixtures were titrated with pronase-treated cells and after keeping for I hour more at room temperature, the degree of hemagglutina-
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tion was examined and the maximum dilution of inhibitor solution showing inhibition was recorded. Controls were set up as described above.
RESULTS
Table 1 represents the hemagglutination profiles of Heterometrus hemolymph with untreated and enzyme-treated human and animal erythrocytes. Heterometrus scorpion hemolymph agglutinated human ABO blood groups equally well and did not show any blood group specificity. Of the animal erythrocytes goat and rabbit RBCs were not agglutinated normally but after pronase treatment agglutination resulted. This indicates that the carbohydrate determinant on these two RBCs became accessible only after partial removal of the glycoprotein coat from the erythrocytes surface. Whereas agglutination of cow and pig RBCs was not observed suggesting the absence of carbohydrate receptor for the hemolymph on these cells. Agglutination of erythrocytes of all species were more strong after pronase treatment owing to the removal of charged groups from the erythrocytes surfaces, and rendered zero by neuraminidase. This indicates that Heterometrus agglutinin shows specificity for sialic acids. However, rat and mouse erythrocytes still persisted agglutination by neuraminidase owing to the presence of 4(7)-O-acetylated sialic acids which are resistant to Vibrio cholerae neuraminidase actlon. In Table 2 typical experiments of crossed-absorption of H. $ranulomanus hemolymph is shown. With Hetrometrus hemolymph mouse and rat Pr- and VCN-treated RBCs exhibited similar agglutination pattern and thus cross-reacted. This indicates that the agglutinin possesses the same carbohydrate binding specificity for these cells. The same specificity also holds true for the rabbit RBCs as the hemolymph after absorption on enzyme-treated mouse and rat erythrocytes did not agglutinate rabbit RBCs. However, hemolymph absorbed on Pr-treated rabbit RBCs was found still active in agglutinating enzyme treated rat and mouse erythrocytes. This suggests that the Heterometrus hemolymph must have adjunct specificity for O-acetylated sialic acids present in these two cells (23). Table 3 shows the amount of sugar (mM) required for inhibition of two agglutination doses of hemolymph. Human RBC agglutination was inhibited by NANA and was twice more effective than NGNA. Mono- and di-O-acetyl-Nacetyl-neuraminic acids were as potent inhibitors as NANA. This suggests that only the N-acetyl group is essential in binding of the hemolymph agglutinins. Halogenated sialic acid was also inhibitor and twice less potent than NANA. Sialyllactose was found to be the most potent inhibitor (6.25 mM) among the mono- and oligosaccharides tested. 2-acetamido-2deoxy-hexoses were non-inhibitors even at 200 mM concentrations. Hexuronic acid viz, D-glucuronic acid is a moderate inhibitor of the hemolymph lectin whereas D-galacturonic acid was non-inhibitor. When certain sialoglycoproteins were examined in the hemagglutination-inhibition reaction (Table 4), glycophorin was found to be most effective inhibitor whereas asialoglycophorin did not inhibit at all. Bird nest glycoprotein was four times less effective than glycophorin and its asialo counterpart did not inhibit indicating the agglutinin to be specific for sialic acid. Similar result was obtained with serum glycoprotein IgD/Fc fraction. Pig erythrocyte mucoid inhibited the hemagglutination and twice less effective than bird nest glycoprotein or IgD/Fc fraction. Blood group substances such as ovarian A and H substances were also inhibitors of which the latter was
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299
TABLE i Hemagglutination Profiles of Heterometrus Hemolymph with Untreated and Enzyme-treated Erythrocytes Enzyme Treatment Untreated
Pronase (Streptomyces griseus)
64 64 64
512 512 512
0 0 0
Sheep
8
32
0
Goat
0
32
0
Rabbit
0
512
0
128
1024
32
32
64
32
Chicken
4
64
0
Pigeon
8
4
0
Buffalo
8
32
0
Duck
2
8
0
Erythrocytes
Human
A B O
Mouse Rat
Neuraminidase (Vibrio cholerae )
TABLE 2 Crossed-Absorption of Heterometrus Hemolymph with Untreated and Enzyme-Treated Erythrocytes Supernatant tested with Heterometrus hemolymph a absorbed with
Mouse
Rat
Rabbit VCN
Pr
VCN
Pr
VCN
Pr
None
64
4
16
8
64
0
Mouse Pr VCN
0 32
16 0
0 8
32 0
0 0
0 0
Pr VCN
0 0
16 4
0 16
16 0
0 0
0 0
Rabbit Pr
64
16
16
16
0
0
Rat
griseus pronase , VCN: Vibrio cholerae neuraminidase; Pr: Streptom . . . . ~ .ces .... . a) diluted i to 4.
twice more effective than the former in the inhibition. Colominic acid, a homopolymer of sialic acid, -NANA-~-(2 ÷ 8)-NANA-, was weak inhibitor like
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LECTIN FROM SCORPION HEMOLYMPH
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TABLE 3 Hemagglutination-inhibition of Heterometrus Hemolymph by Mono- and Oligosaccharides Inhibitors (mM)
Erythrocyte Human (Pronase)
N-acetylneuraminic acid
12.5
N-glycolylne~raminic acid
25.0
Di-O-acetyl-N-acetylneuraminic acid
12.5
O-acetyl-N-acetylneuraminic acid
12.5
Fluoro-N-acetylneuraminic acid
25.0
N-acetyl-neuraminyllactose
6.25
N-acetyl-D-glucosamine
NI
N-acetyl-D-galactosamine
NI
D-glucuronic acid
50
D-galacturonic acid
NI
Figures are minimal concentrations (mM) that inhibit two hemagglutinating doses. NI: No inhibition at tested concentrations upto 200 mM. D-glucose, D-galactose, D-mannose, L-fucose, D-galactosamine HCI, D-glucosamine HCI, lactose, melibiose, D-raffinose did not inhibit at concentrations upto 200 mM.
ovarian A substance (1%). Among the glycoproteins tested peptone A, fetuin and human glycoprotein fraction VI were non-inhibitors. Such ~nusual behaviour of these glycoconjugates is not clear. Hemagglutination titer of the hemolymph remained unchanged when dilutions were carried out in TBS containing 0 . 1 M sodium citrate (64) but reduced in presence of 0 . 1 M dithioerythritol (16) and was completely lost in presence of 0.i M EDTA(0). Titer was also independent of NaCI within the range of 0.15-1.0 M concentration (64). The observed titer was not changed by the addition of I mM Mn 2+ or Mg 2+ (64) but enhanced in presence of Ca2+(128) all as chlorides to the diluent. (The figures in brackets represent titer). The agglutinin was stable for i hour at 56°C, but on heating over an hour completely lost its activity.
DISCUSSION
"The subphylum Chelicerata comprises the classes Merostomata (horseshoe crab) and Arachnida (scorpions, spiders etc.). The Chelicerata exhibits certain homogeneity in the specificity". Hemolymph of all species studied so far contains agglutinins that bind sialoglycoconjugates. The results reported herein ~how that Heterometrus granu!omanus hemolymph also
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TABLE 4 Hemagglutination-inhibition of Heterometrus Hemolymph by Polysaccharides and Glycoproteing Inhibitors (% lw/vl )
Erythrocyte Human (Pronase)
Ovarian A substance
].0
Ovarian H substance
0.5
Bird nest glycoprotein
0.]25
Desialylated bird nest glycoprotein
NI
Glycophorin
0.031
Desialylated glycophorin
NI
Pig erythrocyte mucoid
0.25
Peptone A
NI
IgD/Fc
0.125
Fetuin
NI
Colominic acid
1.0
Human glycoprotein fraction VI
NI
Figures are minimal concentrations (% lwlv I ) that inhibit two hemagg~luti nation doses. NI : no inhibition at tested concentrations (upto I%). Desialylation of glycoproteins was performed by hydrolyzing the glycoproteins with 0.1N H2SO 4 for i hour at 80°C.
binds sialic acid and sialoglyconjugates, since the carbohydrates recognlsed on the RBC membrane are sialic acids because after their removal by VCN the cells were no longer agglutinated. Agglutinins from the hemolymphs of horseshoe crab Limulus polyphemus, saharan scorpion Androctonus australia (19) and the whip scorpion Mastigoproctus g i g a n t e u s (21) exhibit similar agglutination pattern. However, some heterogeneity may be present as observed by crossed-absorption tests. Hemagglutination-inhibition experiments confirm the specificity for sialic acid, and sialoglycoconjugates. Such high specificity of chelicerata lectins for certain carbohydrates has supported the hypothesis that a lack of diversity in specificity possesses the ability to recognise nonself substances. However, this heterogeneity has been proven to be due to molecular structure and specificity. Unlike whip scorpion, saharan scorpion and limulin, Heterometrus agglutinin shows no inhibitory effect by GalNAc or GIdNAc whereas GIcA is inhibitory for both limulin and Heterometrus. The lectins from other species of scorpion studied in other laboratories show specificity for sialic acid but also bind hexuronic acid and N-acety] aminosugars. Detailed study of these primary and secondary specificities in scorpion lectins would be very important to understand possible biologica] functions of these lectins. Sialic acid binding lectins have been detected not only in the chelicerates but also in other arthopods including crustaceans (24,25) insect (26), molluscs (27) and tunicates (28) and appear to be ubiquitous among invertebrates. Attempts have been made to purify and
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characterize this hemolymph lectin to understand the relationship between specificity and molecular structure.
ACKNO~FLEDGEMENTS
The authors wish to thank Prof. C.V.N. Rao, Head of this Department for encouragement in this investigation and Dr. S.B. Dutta, Director, Central Blood Bank, Medical College and Hospital, Calcutta for supplying blood samples.
REFERENCES
i.
NOGUCHI, H. On the multiplicity of the serum hemagglutinins of cold-blooded animals. Zbl. Bakt.~ I. Abt. Ori$., 34, 238, 1903.
2.
BOYD, W . C . 1963.
3.
YEATON, R . W . Invertebrate lectins : I Occurrence. Immunol.'5, 391, 1981.
4.
LIS, H. and SHARON, N. The biochemistry of plant lectins (Phytohemagglutinins). Ann. Rev. Biochem. 42, 541, 1973.
5.
LIENER, I . E . Phytohemagglutinins Physiol. 27, 291, 1976.
6.
MARCHALONIS, J . J . Immunit~ in Evolution. Univ. Press, 1978, p. 316.
7
MARCHALONIS, J. J. and EDELMAN, G . M . Isolation and characterization of a hemagglutinin from Limulus polyphemus. J. Mol. Biol. 32, 453, 1968.
8.
WADSTROM, T., lectins. In mistry. Vol. and New York
9.
COHEN, E. Immunologic observations of the agglutinins of the hemolymph of Limulus polyphemus and Birgus latro. Trans. N. Y. Acad, Sci., 30, 427, 1968.
i0.
PARDOE, G.I., UHLENBRUCK~ G. and BIRD, G.W.G. Studies on some heterophile receptors of the Burkitt EB 2 lymphoma cell. Immunology 18, 73, 1970.
The lectins : their present status.
(phytolectins).
Vox Sang. 8, I,
Dev. Comp.
Ann. Rev. Plant
Cambridge
: Harvard
TRUST, T. J. and BROOKS, D . E . Bacterial surface : Lectins~ Biology, Biochemistry and Clinical BiocheIII T. C. B~g-Hansen and G.A. Spengler (Eds.) Berlin : Walter de Gruyter & Co., 1983, p. 479.
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ii.
ROCHE, A$C., and MONSIGNY, M. Limulin (Limulus polyphemus lectin): Isolation, physicochemical properties, sugar specificlty and mitogenic activity. In: Biomedical Applications of the Horseshoe Crab (Limulidae) E. Cohen, (Ed') New York: Liss, A.R., 1979, p. 603.
12.
SHIMIZU, S., ITO, M. and NIWA, M. Lectins in the hemolymph of Japanese horseshoe crab, Tachypleus tridentatus. Biochim. Biophy s Acta 500, 71, 1977.
13.
SHIMIZU, S., ITO, M., TAKAHASHI, N. and NIWA, M. Purification and properties of lectins from the Japanese horseshoe crab, Tachypleus tridentatus. Pros. Clin. Biol. Res. 29, 625, 1979.
14.
BISHAYEE, S. and DORAI, T. Isolation and characterization of a sialic acid binding lectin (carcinoscorpin) from Indian horseshoe crab C arcingscorpius rotunda cauda. Biochim. Bio~hys. Acta~ 623, 89 1980.
15.
HALL, J.L. and ROWLANDS, JR., D.T. Heterogeneity of lobster agglutinins I. Purification and physicochemical characterization. Biochemistry 13, 821, 1974.
16.
HALL, J.L. and ROWLANDS, JR., D.T. Heterogeneity of lobster agglutinins II. Specificity of agglutinin-erythrocyte binding. Biochemistry 13, 828, 1974.
17.
VASTA, G.R. and COHEN, E. Carbohydrate specificities of Birsus latro (coconut crab) serum lectins. Dev. Comp. Immunol. 8, 197, 1984.
18.
COHEN, E., ILODI, G.H.U., BRAHMI, Z. and MINOWADA, J. The nature of cellular agglutinins of Androctonus australis (saharan scorpion) serum. Dev. Comp. Immunol. 3, 429, 1979.
19.
VASTA, G.R., ILODI, G.H.U., COHEN, E. and BRAHMI, Z. A comparative study on the specificity of Androctonus australis (saharan scorpion) and Limulus polyphemus (horseshoe crab) agglutinins. Dev. Comp. Immunol. 6, 625, 1982.
20.
VASTA, G.R. and COHEN, E. Agglutinins in the hemolymph of the scorpion Centruroides sculpturatus. Fed. Proc. 39, 915, 1979.
21.
VASTA, G.R. and COHEN, E. Sialic acid binding lectins in the Whip scorpion (Mastigoproctus giganteus) serum. J. Invert. Pathol. 43, 333, 1984.
22.
CHATTERJEE, B.P., VAITH, P., CHATTERJEE, S., KARDUCK, D. and UHLENBRUCK, G. Comparative studies of new marker lectins for alkal~ labile and alkali-stable carbohydrate chains in glycoproteins. Int. J. Biochem. 10, 321, 1979.
23.
SCHAUER, R., SHUKLA, A.K., SCHRODER, C. and MULLER, E. The antirecognition function of sialic acids : Studies with erythrocytes and macrophages. Pure Appl. Chem. 56, 907, 1984.
24.
VASTA, G.R., WARR, G.W. and MARCHALONIS, J.J. Serological characterization of Humoral lectins from the freshwater prawn Macrobrachium rosenber~ii. Dev. Comp. Irm~unol. 7, 13, 1983.
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25.
RAVINDRANATH, M.H., HIGA, H.H., COOPER, E.L. and PAULSON, J.C. Purification and characterization of an O-acetyl sialic acid specific lectin from a marine crab Cancer antennarius. J. Biol. Chem. 260, 8850, 1985.
26.
HAPNER, K.D. and JERMYN, M.A. Haemagglutinin activity in the haemolymph of Teleogryl!us commodus (Walker). Insect. Biochem. Ii, 287 1981.
27.
ISHIYAMA, I., TAKATSU, A., GIELEN, W. and UHLENBRUCK, G. An agglutinin from the sea snail Dolabella reacting with a neuraminic acid containing structure. H ematoologia 6, 109, 1972.
28.
VASTA, G.R., WARR, G.W. and MARCHALONIS, J.J. Tunicate lectins : distribution and specificity. C omp. Biochem. physiol. 73B, 887, 1982.
Received: November, 1985 Accepted: March, 1986
SEROLOGICAL CHARACTERIZATION HETEROMETRUS GRANULOMANUS
1986.
OF HUMORAL LECTIN FROM SCORPION HEMOLYMPH*
H. AHMED 1 G. ANJANEYULU 2 and B P.CHATTERJEE 1 iDepartment of Macromolecules, Indian Association for the Cultivation of Science, Jadavpur, Calcutta - 700 032 India 2Department of Zoology, Hindu College, Guntur - 522 002,India
ABSTRACT
A lectin in the hemolymph of Indian scorpion Heterometrus sran ulomanus was detected by agglutination of human and animal erythrocytes. The agglutinating activity was enhanced in presence of Ca2+ ion. The lectin shows specificity for sialic acid like many other sialic acid-specific lectins such as "Limulin", as the agglutination of erythrocytes was completely abolished by treatment with Vibrio cholerae neuraminidase. However, neuraminidase-treated rat and mouse erythrocytes exhibited high titers. This result was substantiated by crossed-absorption test which suggests the adjunct specificity of the Heterometrus lectin for these cells. Hemagglutination-inhibition results of the lectin indicate that sialic acid including its derivatives and sialoglycoconjugates are the inhibitors among which glycophorin was most potent.
INTRODUCTION
Noguchi first observed the presence of hemagglutinin in the coelomic fluid of the horseshoe crab Limulus polyphemus(1). Similar substances, generally now called lectins (2) have gradually been discovered in the hemolymphs and tissue extracts of many invertebrate species, as well as in plants (3-5) and vertebrates (6). Lectins define a class of heat sensitive proteins or glycoproteins which, because of their polyvalent configuration and binding affinity for specific cell surface carbohydrates, can selec-
* Part of this work was presented at the XIIth International Symposium held in Utrecht, July, 1984. 295
Carbohydrate
296
LECTIN FROM SCORPION HEMOLYMPH
Vol. i0, No. 3
tively agglutinate vertebrate erythrocytes. This agghJtination is usually dependent upon the presence of divalent metal ions which apparently stabilize tertiary conformation of lectin molecules (7). They occur as fundamental biological principle in all living systems like in viruses, bacteria and parasites, plants, invertebrates and vertebrates and play an essential role, which may be different in all these systems according to diverse functions. For instance in bacteria they are helpful generally for causing an infection by mediating adhesion to the cell surface through a specific carbohydrate receptor (8). Functional knowledge of invertebrate lectins is rudimentary and many suggestions on them are offered though they are not experimentally verified. Some of these are (i) receptor recognition that is recognition between self and nonself (ii) recognition and removal of aged glycoconjugates and (iii) defence against foreign invaders. Though the study of serum lectins from the chelicerates was pioneered by Noguchi at the begining of this century, complete characterization of Limulus polyphemus hemagglutinin and its specificity for sialic acids was demonstrated by Cohen (9), P~rdoe et al. (i0) and Roche and Monsigny (11). Other Chelicerata studied are Japanese horseshoe crab Tachypleus tridentatus (12,13) and the Indian horseshoe crab Carcinoscorpius rotunda cauda (14). The former exhibits two groups of lectins specific for several N-acylamino sugars and sialic acids whi]e the latter binds sialic acid only. Anti-sialyl lectins have also been found in the Crustacea - the lobster Homarus americanus (15,16) and the coconut crab Birgus latro (17). Within the class Scorpiones, agglutinins were detected in the hemolymph of Saharan scorpion Androctonus australis (18,19) and the Arizona lethal scorpion Centruroides sculpturatus (20), whip scorpion Mastigoproctus giganteus (21).
In the present communication we report different agglutination profiles of Heterometrus hemolymph and its specificity.
MATERIALS AND METHODS
Agglutinins Heterometrus granulomanus scorpions were procured from Andhra Pradesh, India and identified by Zoological Survey of India, Calcutta. The hemolymph from both sexes of living specimens was collected from pedipalp by using 20-gauge needle. The hemolymph was allowed to clot at 25 °C for 1 hour and then centrifuged at 12,000 x g for 30 minutes in the cold. The clear supernatant was preserved at -20 °C until use.
Inhibitors N-acetylneuraminic acid (NANA), N-glycolylneuraminic acid (NGNA), NANA-lactose, N-acetyl-D-glucosamine (GIcNAc), N-acetyl-D-galactosamine (GalNAc), Digalactose, peptone A, IgD/Fc, fetuin, colominic acid, human glycoprotein fraction VI were purchased from Sigma Chemical Co. U.S.A. Ovarian A and H substances, bird nest glycoprotein and pig erythrocyte mucoid were kindly donated by Prof. G. Uhlenbruck, Medical University
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LECTIN FROM SCORPION HEMOLYMPH
Clinic, Cologne, F.R.G. Glycophorin was the kind gift of Dr. A.K. Shukla, Biochemisches Institut, Christian-Albrechts-Universitat, Kiel, F.R.G. Diand mono-O-acetyl-N-acetylneuraminic acid, fluoro-N-acetylneuraminic acid were obtained from Dr. T.K. Ghosh, The Johns Hopkins University, Department of Biology, Baltimore, U.S.A. All other reagents were from Merck or B.D.H.
Erythrocytes Human blood was obtained from healthy donors by vein puncture and was collected in citrate-dextrose solution. Rat, mouse and rabbit blood was obtained either by vein or cardiac puncture. The blood of duck and pigeon was obtained by sacrificing the animals and that of the rest animals was collected from slaughter house. The erythrocytes were washed thrice with saline 0.85% (w/v) and twice with TBS (Tris-buffered saline; I00 mM TrisHCI, 150 mM NaCI, pH 7.6) and suspended at concentrations of 5 x 106 RBCs/ ml in TBS.
Enzymes Pronase P from Streptomyces griseus was purchased from Serva, Heidelberg, F.R.G. Neuramini'dase from Vibrio cholerae (VCN) 500 u/ml was from Behring Werke AG, Marburg, F.R.G. The RBCs were treated with pronase P and VCN according to Chatterjee et al. (22).
Hemagglutination Test This was performed in Takatsy microtiter plates. To two-fold serially diluted Heterometrus hemolymph (25 ~i) in TBS was added equal volumes of the erythrocyte suspension. The plates were incubated at 27 °C for 30 minutes after gentle shaking. Agglutination was read visually. The reciprocal of the highest dilution of the hemolymph showing visible agglutination was recorded as the titer. The experiments were performed in duplicate and controls were set up using TBS instead of hemolymph.
Crossed-absorption Test Diluted hemolymph was mixed with one-third of its volume in washed, packed and enzyme-treated RBCs and incubated for 45 minutes with occssional shaking. After centrifugation at 1000 x g for 5 minutes the supernatant was titrated as described above.
Hemagglutination-inhibition Test All inhibit0rs to be tested were dissolved in TBS at concentrations up to 200 mM for mono- and oligosaccharides and i% (w/v) for po]ysaccharides and glycoproteins adjusted at pH 7.6 with concentrated NaOH and ser~a-fly two-fold diluted in TBS in microtiter p]ates~ Eq,al volumes of diluted hemolymph (Titer 2) were added and incubated for ] hour at room temperature. The mixtures were titrated with pronase-treated cells and after keeping for I hour more at room temperature, the degree of hemagglutina-
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tion was examined and the maximum dilution of inhibitor solution showing inhibition was recorded. Controls were set up as described above.
RESULTS
Table 1 represents the hemagglutination profiles of Heterometrus hemolymph with untreated and enzyme-treated human and animal erythrocytes. Heterometrus scorpion hemolymph agglutinated human ABO blood groups equally well and did not show any blood group specificity. Of the animal erythrocytes goat and rabbit RBCs were not agglutinated normally but after pronase treatment agglutination resulted. This indicates that the carbohydrate determinant on these two RBCs became accessible only after partial removal of the glycoprotein coat from the erythrocytes surface. Whereas agglutination of cow and pig RBCs was not observed suggesting the absence of carbohydrate receptor for the hemolymph on these cells. Agglutination of erythrocytes of all species were more strong after pronase treatment owing to the removal of charged groups from the erythrocytes surfaces, and rendered zero by neuraminidase. This indicates that Heterometrus agglutinin shows specificity for sialic acids. However, rat and mouse erythrocytes still persisted agglutination by neuraminidase owing to the presence of 4(7)-O-acetylated sialic acids which are resistant to Vibrio cholerae neuraminidase actlon. In Table 2 typical experiments of crossed-absorption of H. $ranulomanus hemolymph is shown. With Hetrometrus hemolymph mouse and rat Pr- and VCN-treated RBCs exhibited similar agglutination pattern and thus cross-reacted. This indicates that the agglutinin possesses the same carbohydrate binding specificity for these cells. The same specificity also holds true for the rabbit RBCs as the hemolymph after absorption on enzyme-treated mouse and rat erythrocytes did not agglutinate rabbit RBCs. However, hemolymph absorbed on Pr-treated rabbit RBCs was found still active in agglutinating enzyme treated rat and mouse erythrocytes. This suggests that the Heterometrus hemolymph must have adjunct specificity for O-acetylated sialic acids present in these two cells (23). Table 3 shows the amount of sugar (mM) required for inhibition of two agglutination doses of hemolymph. Human RBC agglutination was inhibited by NANA and was twice more effective than NGNA. Mono- and di-O-acetyl-Nacetyl-neuraminic acids were as potent inhibitors as NANA. This suggests that only the N-acetyl group is essential in binding of the hemolymph agglutinins. Halogenated sialic acid was also inhibitor and twice less potent than NANA. Sialyllactose was found to be the most potent inhibitor (6.25 mM) among the mono- and oligosaccharides tested. 2-acetamido-2deoxy-hexoses were non-inhibitors even at 200 mM concentrations. Hexuronic acid viz, D-glucuronic acid is a moderate inhibitor of the hemolymph lectin whereas D-galacturonic acid was non-inhibitor. When certain sialoglycoproteins were examined in the hemagglutination-inhibition reaction (Table 4), glycophorin was found to be most effective inhibitor whereas asialoglycophorin did not inhibit at all. Bird nest glycoprotein was four times less effective than glycophorin and its asialo counterpart did not inhibit indicating the agglutinin to be specific for sialic acid. Similar result was obtained with serum glycoprotein IgD/Fc fraction. Pig erythrocyte mucoid inhibited the hemagglutination and twice less effective than bird nest glycoprotein or IgD/Fc fraction. Blood group substances such as ovarian A and H substances were also inhibitors of which the latter was
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TABLE i Hemagglutination Profiles of Heterometrus Hemolymph with Untreated and Enzyme-treated Erythrocytes Enzyme Treatment Untreated
Pronase (Streptomyces griseus)
64 64 64
512 512 512
0 0 0
Sheep
8
32
0
Goat
0
32
0
Rabbit
0
512
0
128
1024
32
32
64
32
Chicken
4
64
0
Pigeon
8
4
0
Buffalo
8
32
0
Duck
2
8
0
Erythrocytes
Human
A B O
Mouse Rat
Neuraminidase (Vibrio cholerae )
TABLE 2 Crossed-Absorption of Heterometrus Hemolymph with Untreated and Enzyme-Treated Erythrocytes Supernatant tested with Heterometrus hemolymph a absorbed with
Mouse
Rat
Rabbit VCN
Pr
VCN
Pr
VCN
Pr
None
64
4
16
8
64
0
Mouse Pr VCN
0 32
16 0
0 8
32 0
0 0
0 0
Pr VCN
0 0
16 4
0 16
16 0
0 0
0 0
Rabbit Pr
64
16
16
16
0
0
Rat
griseus pronase , VCN: Vibrio cholerae neuraminidase; Pr: Streptom . . . . ~ .ces .... . a) diluted i to 4.
twice more effective than the former in the inhibition. Colominic acid, a homopolymer of sialic acid, -NANA-~-(2 ÷ 8)-NANA-, was weak inhibitor like
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TABLE 3 Hemagglutination-inhibition of Heterometrus Hemolymph by Mono- and Oligosaccharides Inhibitors (mM)
Erythrocyte Human (Pronase)
N-acetylneuraminic acid
12.5
N-glycolylne~raminic acid
25.0
Di-O-acetyl-N-acetylneuraminic acid
12.5
O-acetyl-N-acetylneuraminic acid
12.5
Fluoro-N-acetylneuraminic acid
25.0
N-acetyl-neuraminyllactose
6.25
N-acetyl-D-glucosamine
NI
N-acetyl-D-galactosamine
NI
D-glucuronic acid
50
D-galacturonic acid
NI
Figures are minimal concentrations (mM) that inhibit two hemagglutinating doses. NI: No inhibition at tested concentrations upto 200 mM. D-glucose, D-galactose, D-mannose, L-fucose, D-galactosamine HCI, D-glucosamine HCI, lactose, melibiose, D-raffinose did not inhibit at concentrations upto 200 mM.
ovarian A substance (1%). Among the glycoproteins tested peptone A, fetuin and human glycoprotein fraction VI were non-inhibitors. Such ~nusual behaviour of these glycoconjugates is not clear. Hemagglutination titer of the hemolymph remained unchanged when dilutions were carried out in TBS containing 0 . 1 M sodium citrate (64) but reduced in presence of 0 . 1 M dithioerythritol (16) and was completely lost in presence of 0.i M EDTA(0). Titer was also independent of NaCI within the range of 0.15-1.0 M concentration (64). The observed titer was not changed by the addition of I mM Mn 2+ or Mg 2+ (64) but enhanced in presence of Ca2+(128) all as chlorides to the diluent. (The figures in brackets represent titer). The agglutinin was stable for i hour at 56°C, but on heating over an hour completely lost its activity.
DISCUSSION
"The subphylum Chelicerata comprises the classes Merostomata (horseshoe crab) and Arachnida (scorpions, spiders etc.). The Chelicerata exhibits certain homogeneity in the specificity". Hemolymph of all species studied so far contains agglutinins that bind sialoglycoconjugates. The results reported herein ~how that Heterometrus granu!omanus hemolymph also
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TABLE 4 Hemagglutination-inhibition of Heterometrus Hemolymph by Polysaccharides and Glycoproteing Inhibitors (% lw/vl )
Erythrocyte Human (Pronase)
Ovarian A substance
].0
Ovarian H substance
0.5
Bird nest glycoprotein
0.]25
Desialylated bird nest glycoprotein
NI
Glycophorin
0.031
Desialylated glycophorin
NI
Pig erythrocyte mucoid
0.25
Peptone A
NI
IgD/Fc
0.125
Fetuin
NI
Colominic acid
1.0
Human glycoprotein fraction VI
NI
Figures are minimal concentrations (% lwlv I ) that inhibit two hemagg~luti nation doses. NI : no inhibition at tested concentrations (upto I%). Desialylation of glycoproteins was performed by hydrolyzing the glycoproteins with 0.1N H2SO 4 for i hour at 80°C.
binds sialic acid and sialoglyconjugates, since the carbohydrates recognlsed on the RBC membrane are sialic acids because after their removal by VCN the cells were no longer agglutinated. Agglutinins from the hemolymphs of horseshoe crab Limulus polyphemus, saharan scorpion Androctonus australia (19) and the whip scorpion Mastigoproctus g i g a n t e u s (21) exhibit similar agglutination pattern. However, some heterogeneity may be present as observed by crossed-absorption tests. Hemagglutination-inhibition experiments confirm the specificity for sialic acid, and sialoglycoconjugates. Such high specificity of chelicerata lectins for certain carbohydrates has supported the hypothesis that a lack of diversity in specificity possesses the ability to recognise nonself substances. However, this heterogeneity has been proven to be due to molecular structure and specificity. Unlike whip scorpion, saharan scorpion and limulin, Heterometrus agglutinin shows no inhibitory effect by GalNAc or GIdNAc whereas GIcA is inhibitory for both limulin and Heterometrus. The lectins from other species of scorpion studied in other laboratories show specificity for sialic acid but also bind hexuronic acid and N-acety] aminosugars. Detailed study of these primary and secondary specificities in scorpion lectins would be very important to understand possible biologica] functions of these lectins. Sialic acid binding lectins have been detected not only in the chelicerates but also in other arthopods including crustaceans (24,25) insect (26), molluscs (27) and tunicates (28) and appear to be ubiquitous among invertebrates. Attempts have been made to purify and
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characterize this hemolymph lectin to understand the relationship between specificity and molecular structure.
ACKNO~FLEDGEMENTS
The authors wish to thank Prof. C.V.N. Rao, Head of this Department for encouragement in this investigation and Dr. S.B. Dutta, Director, Central Blood Bank, Medical College and Hospital, Calcutta for supplying blood samples.
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Received: November, 1985 Accepted: March, 1986