Purification, some properties of a D-galactose-binding leaf lectin from Erythrina indica and further characterization of seed lectin

Biochimie 84 (2002) 1035–1043 www.elsevier.com/locate/biochi

Purification, some properties of a D-galactose-binding leaf lectin from Erythrina indica and further characterization of seed lectin Emadeldin H.E. Konozy a,*, Ranjana Mulay b, Vitor Faca c, Richard John Ward a,d, Lewis Joel Greene a,c, Maria Cristina Roque-Barriera a, Sushma Sabharwal e, Shobhana V. Bhide e a

Departamento de Biologia Celular, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, 14049-900 Ribeirão Preto, Brazil b Department of Chemistry, Ahmednagar College, Ahmednagar, India c Centro de Química de Proteínas, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Universidade de São Paulo, 14049-900 Brazil d Departamento de Química, FFCLRP, Universidade de São Paulo, 14049-900 Ribeirão Preto, Brazil e Department of Chemistry, Centre for advanced studies in Chemistry, University of Pune, Pune 411007, India Received 26 March 2002; accepted 5 July 2002

Abstract Lectin from a leaf of Erythrina indica was isolated by affinity chromatography on Lactamyl-Seralose 4B. Lectin gave a single band in polyacrylamide gel electrophoresis (PAGE). In SDS-gel electrophoresis under reducing and non-reducing conditions Erythrina indica leaf lectin (EiLL) split into two bands with subunit molecular weights of 30 and 33 kDa, whereas 58 kDa was obtained for the intact lectin by gel filtration on Sephadex G-100. EiLL agglutinated all human RBC types, with a slight preference for the O blood group. Lectin was found to be a glycoprotein with a neutral sugar content of 9.5%. The carbohydrate specificity of lectin was directed towards D-galactose and its derivatives with pronounced preference for lactose. EiLL had pH optima at pH 7.0; above and below this pH lectin lost sugar-binding capability rapidly. Lectin showed broad temperature optima from 25 to 50 °C; however, at 55 °C EiLL lost more than 90% of its activity and at 60 °C it was totally inactivated. The pI of EiLL was found to be 7.6. The amino acid analysis of EiLL indicated that the lectin was rich in acidic as well as hydrophobic amino acids and totally lacked cysteine and methionine. The N-terminal amino acids were Val-Glu-Thr-IIe-Ser-Phe-Ser-PheSer-Glu-Phe-Glu-Ala-Gly-Asn-Asp-X-Leu-Thr-Gln-Glu-Gly-Ala-Ala-Leu-. Chemical modification studies of both EiLL and Erythrina indica seed lectin (EiSL) with phenylglyoxal, DEP and DTNB revealed an absence of arginine, histidine and cysteine, respectively, in or near the ligand-binding site of both lectins. Modification of tyrosine with NAI led to partial inactivation of EiLL and EiSL; however, total inactivation was observed upon NBS-modification of two tryptophan residues in EiSL. Despite the apparent importance of these tryptophan residues for lectin activity they did not seem to have a direct role in binding haptenic sugar as D-galactose did not protect lectin from inactivation by NBS. © 2002 E´ditions scientifiques et médicales Elsevier SAS and Société française de biochimie et biologie moléculaire. All rights reserved. Keywords:Erythrina indica; Leguminasae; Lectin; Characterization; Chemical modification; Tryptophan

1. Introduction Lectins are sugar stereospecific binding proteins or glycoproteins capable of reversibly agglutinating cells. They have Abbreviations: EiSL, Erythrina indica seed lectin; EiLL, Erythrina indica leaf lectin; EvL, Erythrina variegata lectin; EveL, Erythrina velutina forma aurantiaca lectin; EcristL, Erythrina cristagalli lectin; EhL, Erythrina humeana lectin; EpL, Erythrina perrieri lectin; EfL, Erythrina flabelliformis lectin; FrA, Fraction A; PBS, Phosphate buffer saline; DEP, Diethylpyrocarbonate; NAI, N-acetylimidazole; NBS, N-bromosuccinimide; HNBBr, 2-hydroxy-5-nitrobenzyl bromide; DTNB, bis-dithionitrobenzoic acid; Trp, Tryptophan; HAU, Hemagglutunation unit. * Corresponding author. Fax: +1-510-663-6535 E-mail address: [email protected] (E.H.E. Konozy)

attracted great interest because of their effects on various biological systems such as cell agglutination [1], homing of leukocytes [2], induction of mitosis in lymphocytes, interferon c (IFN c) and cytotoxicity [3]. In practical terms, lectins are excellent macromolecular tools for the study of carbohydrate architecture and dynamics at the cell surface during cell division, differentiation and malignancies and, for isolation and characterization of glycoconjugates [4]. Lectin affinity chromatography has been utilized in the assays of many different enzymes, including glycosyltransferases and glycosidases [5]. It is assumed that lectins play a fundamental biological role in plants since they are found in organs and tissues of many different species [6]. Though

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lectins have been extensively studied from plant seeds, they are also detected, yet, in low quantities, in other vegetative tissues such as root, stem, leaf, bark, etc. Some may possess strong homologies with seed lectins (the current study), whereas, other may display unusual carbohydrate binding in their erythrocytes agglutination characteristics [7,8]. Despite the ubiquitous nature of lectins in plant system, their physiological significance remains speculative. Many galactose N-acetylgalactosamine specific lectins have been isolated from various species of the genus Erythrina, which is a family of deciduous leguminous trees and shrubs occurring throughout the tropics and subtropics, their carbohydrate specificity is directed towards D-galactose [9–14]. Among them Erythrina corallodendron seed lectin has been well investigated to a molecular as well as a crystallographic level in complex with mono or disaccharides [15,16]. Erythrina indica is another member of this family in which the seed lectin has been isolated and partially characterized [12,13], Erythrina indica seed lectin (EiSL) is a heterodimer of 30 and 33 kDa, galactose specific. In all of the previously cited studies on the Erythrina genus, characterization of the vegetative tissues lectins is lagging far behind. We have detected hemagglutinating activity in the bark and leaf of Erythrina indica and here we report on the isolation and characterization of leaf lectin. To our knowledge this is the first report on a vegetative tissue lectin from the genus Erythrina.

collected by centrifugation was dehydrated with cold acetone to get a lipid free acetone-dried powder which was subjected to 0.145 M NaCl extraction for 4 h with continuous stirring. The filtrate, called saline extract, was subjected to 80% (NH4)2SO4 fractionation. The precipitate was dissolved in 0.145 M NaCl and dialyzed against the same till free of (NH4)2SO4, it was denoted Fraction A (FrA) and was used for purification of EiLL and EiSL using Lactamyl-Serlaose 4B affinity matrix [18]. Seeds of Erythrina indica were processed for FrA as shown above for leaves. 2.2. Preparation of Lactamyl-Seralose 4B affınity matrix Sixty grams of AH-Seralose 4B were suspended in 40 ml 2 M NaOH and 9 ml epichlorohydrin. The mixture was incubated at 40 °C with shaking for 4 h, and then washed with distilled water to obtain epoxy activated Seralose 4B to which 1.5 volume of ammonium solution was added and reincubated at 40 °C with continuous shaking for 90 min. The aminated gel was thoroughly washed with distilled water. To this amino-Seralose 45 ml of 0.2 M H2PO4, 0.765 g of NaCNBH3 and 1.56 g lactose were added and the suspension was incubated at room temperature (25–28 °C) for 21 days with intermittent shaking. Gel beads were washed with 0.145 M NaCl and preserved in the same buffer in 0.05% (w/v) NaN3 till further use. 2.3. Isolation of EiLL and EiSL

2. Materials and methods Molecular weight markers, ampholines (pH 3–10), all sugars, diethylpyrocarbonate (DEP), N-acetylimidazole (NAI), N-bromosuccinimide (NBS), 2-hydroxy-5nitrobenzyl bromide (HNB-Br), bis-dithionitrobenzoic acid (DTNB) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Standard amino acids (Standard H) were bought from Pierce Chemical Co., Rockford, Illinois. AH-Seralose 4B (product of SRL Co., Bombay, India, similar to Agarose 4B) was generously gifted by Dr. G.S. Murthy, IISC, Bangalore, India. All other reagents were of analytical grade. 2.1. Preparation of Erythrin indica leaf/seed extract Preparation of Erythrina indica leaf/seed extract was carried out essentially as described by Sawhney and Bhide [17]. In brief, 100 g young leaves/seeds of Erythrina indica were obtained from trees on the campus of University of Pune, India. The leaves were deveined and homogenized in a blender with 0.145 M NaCl (500 ml per 100 g of deveined leaves, to get a slurry). The homogenate was mixed with 1-butanol (40 ml per 100 ml of slurry) and stirred at 4 °C for 4 h. The butanol layer was removed by centrifugation. To the homogenate were added equal volumes (50% saturation) of cold acetone under stirring at 4 °C. The resulting precipitate

Lactamyl-Seralose 4B was packed into (1.5 × 30 cm) column pre-equilibrated with 0.145 M NaCl; approximately 100 mg protein of FrA (leaves or seeds extract) were loaded on the column and the unretained protein was washed then with 0.145 M NaCl, and checked for lectin activity. The retained lectin was eluted with 0.2 M lactose in 0.145 M NaCl. The peak fractions detected by hemagglutination were pooled, exhaustively dialyzed against distilled water, lyophilized to dryness and preserved at –10 °C till further use. 2.4. Hemagglutinating activity assay Hemagglutinating activity was determined in wells of microtiter plates in a final volume of 70 µl. Each well contained 50 µl of lectin solution and 20 µl of 4% (v/v) suspension of erythrocytes. Agglutination was assessed after incubation for 1 h at room temperature, and agglutinating activity was expressed as units, namely, the reciprocal of the highest dilution that gave a positive result. The specific hemagglutinating activity was defined as unit per mg protein. The inhibitory effect of sugars was measured by mixing serial dilutions of the inhibitor with four hemagglutinating units of the lectin before addition of erythrocytes and determining lowest concentration giving full inhibition of agglutination.

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2.5. Effect of temperature and pH on EiLL activity

2.10. Amino acid analysis

The influence of temperature on lectin activity was determined as described by Akev and Can [19]. The activity of lectin at various pH values was assayed using aliquots of lectin solutions (1 mg/ml) incubated at different pH values (3–10) for 1 h at room temperature (25–27 °C). Lectin samples were adjusted to pH 7.0 by addition of 0.1 N NaOH or 0.1 N HCl and finally assayed for hemagglutinating activity.

Amino acid analyses were performed using the phenylthiocarbamyl derivatives method [25] after acid hydrolysis with 6 N HCl containing 1% phenol in the vapor phase, at 110 °C for 22 h. A mixture containing 2.5 nmol amino acid was derivatized each day, and 100 pmol was used as standard.

2.6. Carbohydrate content of EiLL This was determined by the phenol–sulfuric acid method of Dubois et al. [20] using D-mannose as the standard.

2.11. N-terminal determination The N-terminal determination was performed using Procise sequenator model 491 (Perkin–Elmer-Applied Biosystem Division, Foster City, California) using either gas-phase or pulse liquid chemistry with on-line identification of phenylthiohydantoin derivatives. 2.12. Protein estimation

2.7. Isoelectric focusing Electrofocusing on a 10% polyacrylamide gel was carried out on Pharmacia Phast System instrument, using Pharmacia broad range IEF calibration kit (pH 3–10). Seven micrograms of the lectin sample were applied and the gel was stained with Coomassie brilliant blue R-250. 2.8. Polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE Polyacrylamide gel electrophoresis (PAGE) and SDSPAGE for the purified lectin were carried as described [21,22], respectively. Gels were silver stained [23].

Protein concentrations were determined according to Lowry et al. [26] using BSA as the standard. 2.13. Chemical modification studies The modifications of tryptophan residues were carried out using NBS [27] and HNB-Br [28]. DTNB was used for modification of cysteine [29]. N-acetyl immidazole was used for modification of tyrosine residues [30]. Phenylglyoxal was used for the modification of arginine residues [31], whereas diethylpyrocarbonate (DEP) was used for modification of histidine [32]. The protocols followed for modification of these residues are shown in Table 1 . 2.14. NBS modification of EiSL

2.9. Immunodiffusion studies EiSL (5 mg) was emulsified with 0.5 ml of Freund’s complete adjuvant and injected subcutaneously into a rabbit. A booster schedule of three doses with 10-day intervals between each dose was followed. Serum from the blood of an immunized rabbit was separated by 50% (NH4)2SO4 saturation to precipitate the IgG. The immunological crossreactivity between EiSL and EiLL for EiLL antibodies was tested by performing Ouchterlony’s double diffusion method [24].

Oxidation of EiSL with NBS was carried out according to the method of Spande and Witkop [27] at room temperature. To a solution of EiSL (1 mg/ml) in 50 mM sodium acetate buffer, pH 6.0, taken in a quartz cell was added an aliquot (10 µl) of 10 mM NBS in water with rapid mixing. After 10 min, the absorbance at 280 nm was recorded. Addition of the reagent was continued until 70 µl of the reagent were added. The number of oxidized tryptophans was calculated using the molar extinction coefficient for tryptophan (5500 per M/cm). After every NBS addition an aliquot was re-

Table 1 Chemical modification of arginine, tryptophan, histidine, cysteine and tyrosine

Amino acid Buffer Temp (°C) Time (min) Concentration (mM) Solvent

Phenylglyoxal

NBS

Chemical modifier HNB-Br

Arginine Sodium bicarbonate 0.1 M, pH 8.0 30 60 10 Water

Tryptophan Acetate buffer, 0.05 M, pH 6.0 30 60 10 Water

Tryptophan Acetate buffer, 0.05 M, pH 3.0. 30 60 10 Dry acetone

DEP

DTNB

NAI

Histidine PBS, 0.1 M, pH 7.2

Cysteine Phosphate buffer, 0.1 M, pH 8.0 30 60 10 Water

Tyrosine Tris–HCl, 0.05 M, pH 8.0 30 60 10 Water

30 60 20 Absolute alcohol

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moved and excess reagent was quenched by addition of 20 mM tryptophan solution. The lectin sample was dialyzed exhaustively against BPS and tested for its hemagglutinating activity. Modification of tryptophan with HNB-Br was performed essentially according to procedures of Horton and Koshland [28] as shown in Table 1. 2.15. Modification of tyrosine, histidine, cysteine and arginine Purified EiSL fractions were preincubated with modifiers at defined conditions (Table 1). The samples of modified lectin were dialyzed exhaustively to remove excess modifiers, lyophilized to dryness and hemagglutinating activity was assayed. 2.16. Residual activity in chemically modified protein Percentage residual activities were calculated using the native and unmodified protein as control possessing 100% activity. 2.17. Substrate protection The effect of substrate protection was studied by preincubating EiSL with excess of lactose followed by treatment with NBS. 2.18. Fluorescence spectroscopy Fluorescence spectra of native and tryptophan modified EiSL were recorded on SLM Amico-8100 spectrofluorimeter at 25 °C. The samples were excited at 295 nm and the emission spectra were recorded between 300 and 400 nm. Quenching of tryptophan fluorescence was measured after titrating 100 µg EiSL with different concentrations of lactose (5–100 mM).

3. Results and discussion

Fig. 1. Affinity chromatography of Erythrina indica on Lactamyl-Seralose 4B. Fifteen milligrams of dialyzed ammonium sulfate dissolved precipitate (FrA) were applied to a 2 × 15 cm column equilibrated with 0.145 M NaCl. Elution was done with 0.2 M lactose.

Since lactose was a stronger inhibitor than D-galactose, Lactamyl-Seralose 4B affinity matrix was chosen for purification of this hemagglutinating activity from leaf extract. Aqueous extract of Erythrina indica leaves was subjected to 80% (NH4)2SO4 fractionation to obtain FrA. This step led to a purification of lectin activity of 10.2-fold, with specific activity 7640 HAU per mg. FrA was subjected to LactamylSeralose 4B affinity chromatography. The affinity resin retained all hemagglutinating activity in FrA, which was eluted by 0.2 M lactose in 0.145 M NaCl (Fig. 1 ). No lectin activity was detected in the column effluent. The protein eluted from the affinity matrix with 0.2 M lactose achieved 49.6-fold purification with an increment on the specific activity up to 37 200 HAU per mg, Table 2 . The obtained lectin was further purified by gel filtration on Sephadex G-100, which resulted in a single symmetrical peak (Fig. 2 ). Upon subjecting the pooled, dialyzed and concentrated protein to PAGE pH 8.3 a single, however, broad band was obtained Fig. 3a . 3.1. Native and subunit structure of EiLL

Crude extract of Erythrina indica leaves contains a hemagglutinating activity that agglutinated all human blood cells with slightly more activity towards the O blood type at a minimum concentration of 10 µg (data not shown). This hemagglutinating activity was inhibited by haptenic sugar D-galactose and lactose (the later being four times stronger).

The MW of the intact EiLL was examined by gel filtration on Sephadex G-100; the result indicated 58 kDa for EiLL. The lectin was further analyzed by SDS-PAGE for revealing its subunit molecular mass (es) in the presence and absence of 2-mercaptoethanol. As depicted in Fig. 3c, EiLL

Table 2 Purification summary of EiLL Fraction

Total protein (mg)

Total unit activity HAU

Specific activity (HAU per mg–1)

Purification (fold)

Saline extract* Fraction A Affinity purified fraction

900 67 1.9

25 600 10 240 40 960

750 7640 37 200

1.0 10.18 49.6

* Saline extract was obtained from homogenizing ca 300 g butanol-defatted leaves.

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independently (Fig. 3a, b) which, may indicate either a difference in a few amino acids resulting in different charge separation or difference in glycosylation pattern. Lamb et al. have shown that Griffonia simplicifolia leaf lectin (GS-II) resembles seed GS-II lectin with respect to biological properties and amino acid composition; however, they differ in their subunit molecular weights, electrophoretic mobilities and isoelectric points. These variations were attributed to a probable different glycosylation points [33].

3.2. Other physiochemical properties EiLL is a glycoprotein with 9.5% neutral sugar content. The optical absorbance coefficient (A 1%280 nm) is 12.5. Its isoelectric point (pI) is 7.6.

3.3. Sugar inhibition and blood group specificity Fig. 2. Gel filtration of purified EiLL on Sephadex G-100. Six hundred micrograms were loaded on a column (2 × 40 cm) equilibrated with 0.145 M NaCl. Fractions of 3 ml were collected at a rate of 5 ml/h.

yielded two adjacent bands with molecular masses of 33 and 30 kDa in the presence and absence of 2-mercaptoethanol indicating the absence of a disulphide bond holding the chains together. As far as subunit molecular mass is concerned EiLL seems to be identical to the counterpart seed lectin (EiSL); however, upon running simultaneous PAGE pH 8.3 for both EiLL and EiSL, the two lectins migrated

EiLL agglutinated erythrocytes of all blood types, A, B, AB and O, with a slight preference for the O blood group (data not shown), like all other Erythrina lectins showing no human blood group specificity [9–14]. Among the various carbohydrates tested, the inhibition of hemagglutination showed that the activity was confined to D-galactose and related derivatives. This inhibitory effect on lectin activity gives further support to classification of this lectin as a member of galactose specific hemagglutinin (Table 3 ). Lactose was the most potent inhibitor for EiLL hemagglutinating activity (Minimum Inhibitory Concentration (MIC) = 1 mM) followed by N-acetyllactosamine and N-acetylgalactosamine MIC = 2 mM). Whereas maximum inhibitory concentration was obtained with p-nitrophenyl a- and b-galactopyranoside (9 mM). Table 3 Carbohydrate inhibition of agglutination by EiLL

Fig. 3. Polyacrylamide gel electrophoresis of EiLL Lane a and b: Simultaneous electrophoresis pH 8.6 run for EiSL and EiLL respectively, 10 µg were loaded at each well and gel was Coomassie blue stained. Lane c: SDS-PAGE of EiLL. Five micrograms were loaded and gel was silver stained. Protein markers used were: Phosphorilase (97 kDa), BSA (66 kDa), Egg albumin (45 kDa) and Carbonic anhydrase (29 kDa).

Carbohydrate

Minimal inhibitory concentration (mM)*

N-acetyllactosamine Lactose D-Galactose N-acetyl-D-galactosamine Methyl-a-D-galactopyranoside Methyl-b-D-galactopyranoside Galactosamine p-Nitrophenyl-␣-D-galactopyranoside p-nitrophenyl-b-D-galactopyranoside Melibiose Raffinose D-mannose D-glucose

2 1 5 2 7 7 3 9 9 3 3 N.I N.I

* Minimal concentration of sugar that brings about complete inhibition of four doses of EiLL. N.I, Sugar not inhibitory until 100 mM.

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p-Nitrophenyl a- and b-galactopyranosides exhibited similar inhibitory effect on EiLL activity (9 mM), Bhattacharyya et al. have reported similar results for Erythrina arborescens seed lectin [13], whereas Lis et al. have shown different inhibitory effects for p-nitrophenyl a- and b-galactopyranosides when tested for nine different Erythrina species lectins [14]. Methyl-a-D-galactopyranoside and methyl-b-D-galactopyranoside inhibited hemagglutination activity of EiLL to a similar extent. These results suggested that EiLL does not differentiate between a- and b-galactoside since both sugars inhibited lectin activity to a similar degree. The strong inhibition by lactose, N-acetyllactosamine and melibiose over galactose indicated that EiLL possesses an extended binding site [14]. The importance of C-4 hydroxyl group orientation is indicated by the failure of mannose or glucose to act as inhibitor. As a whole the MIC values resemble those reported for other Erythrina lectins [9–14]. 3.4. Effect of pH and thermal stability on EiLL EiLL was stable over a wide range of pH values between pH 6.0 and 8.0 with an activity maximal around pH 7.0. Below pH 6.0 and above pH 9.0 lectin showed no activity and above pH 8.0 it rapidly lost its activity. EiLL is thermally stable over a wide range of temperatures between 25 and 50 °C; however, after 50 °C, EiLL started losing activity very rapidly. At 60 °C the lectin lost almost all of its activity. 3.5. Amino acid composition of EiLL Like other Erythrina species lectins, EiLL is rich in acidic and hydroxyl amino acids, however, lacking cysteine. Bhattacharyya et al. [13] have reported six methionine residues per mole of EiSL, but in the present investigation we did not detect any methionine residues in EiLL. EiLL also showed higher content of hydrophobic amino acids such as glycine, alanine, valine and leucine. A summary of EiLL amino acid composition is given in Table 4 .

Table 4 Amino acid composition of EiLL Amino acid Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys IIe Leu Phe Lys Trp TOTAL

Mol % 11.8 10.7 7.0 8.5 1.9 2.6 8.2 9.8 7.4 3.6 6.6 – – 6.0 6.3 5.3 4.4 ND 100

Residues (per M–1)* 69 63 41 50 11 15 48 57 44 21 39 – – 35 37 31 26 ND 588

* The values are based on the assumption of molecular weight of 63 kDa with 9.5% carbohydrate. N.D. Not determined.

3.6. N-terminal determination The N-terminal amino acid sequence determined by Edman’s degradation method was Val-Glu-Thr-IIe-Ser-PheSer-Phe-Ser-Glu-Phe-Glu-Ala-Gly-Asn-Asp-X-Leu-Thr-GlnGlu-Gly-Ala-Ala-Leu-. Horesji et al. [12] have reported the following N-terminal sequence for EiSL Val-Glu-Val-Leu(Phe)-Phe-(Ala)-Phe-; however, when we redetermined the N-terminal for EiSL we obtained the following sequence Val-Glu-Thr-IIe-Ser-Phe-Ser-Phe-Ser-Glu-Phe-Glu-, which shares 100% homology with counterpart leaf lectin. The result of Horesji et al. is likely to be incorrect. EiLL N-terminal amino acid sequence up to position number 12 shares 100% homology with EvL, EcorL, EhL, EfL and EpL (See Table 5). Position number 17 in EiLL N-terminal did not provide a PTH amino acid which is probably a glycosylation point at Asn. In EcorL, it has been reported that the same position (amino acid no. 17) is a glycosylation point at Asn

Table 5 Amino terminal of EiLL in comparison with EiSL and other Erythrina species lectins Erythrina lectin

N-terminal residues

References

EiLL EiSL EveL EvL EcristL EcafL EcorL EhL EpL EfL

Val-Glu-Thr-lle-Ser-Phe-Ser-Phe-Ser-Glu-Phe-GluVal-Glu-Thr-lle-Ser-Phe-Ser-Phe-Ser-Glu-Phe-GluVal-Glu-Thr-lle-Pro-Phe-SerVal-Glu-Thr-lle-Ser-Phe-Ser-Phe-Ser-Glu-Ala-GlyVal-Glu-Thr-lle-Ser-Phe-Ser-Phe-Ser-Glu-Phe-ProVal-Glu-Thr-lle-Ser-Phe-Ser-Phe-Ser-Glu-Phe-ProVal-Glu-Thr-lle-Ser-Phe-Ser-Phe-Ser-Glu-Phe-GluVal-Glu-Thr-lle-Ser-Phe-Ser-Phe-Ser-Glu-Phe-GluVal-Glu-Thr-lle-Ser-Phe-Ser-Phe-Ser-Lys-Phe-GluVal-Glu-Thr-lle-Ser-Phe-Ser-Phe-Ser-Glu-Phe-Glu

CS CS [34] [35] [14] [14] [14] [14] [14] [14]

CS, Current study.

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Fig. 4. Immuno-cross-reaction of EiLL (denoted by E) and EiSL (denoted by S) against EiSL anti-sera (Central well, denoted AbS). Fifty micrograms EiSL and EiLL were placed in the peripheral wells (according to denotations) and 100 µg of EiSL antibodies (AgG) was placed in the central well. Plate was left over-night for diffusion.

[36]. Amino terminal of EiLL in comparison with EiSL and other Erythrina species lectins is depicted in Table 5. It is evident from the table that the homology between EiLL and EiSL is not confined to these two lectins, but may cover the entire genus. 3.7. Cross-reactivity of EiLL with anti-EiSL antibodies The degree of immunological homology between EiLL and EiSL was assessed by Ouchterlony’s double diffusion method [24]. Fig. 4 shows the typical results for the study. It is quite clear that both lectins share common epitopes. The antibodies raised against EiSL formed a single precipitin line with both EiLL and EiSL that grew towards each other and fused to give a single hexagonal shape of complete identity. These results are not surprising as many legume lectins are shown to share varying degrees of identity, which is explained by a common ancestral gene [37,38] 3.8. Chemical modification studies A common strategy for identifying the amino acid residues essential for the biological function of a protein is to treat the protein with specific affinity modifying reagents, which is a common methodology known for several years [39–42]. This investigation provides clues about amino acids involved in catalysis or biological activity. Phenylglyoxal, DEP and DNB-Br did not produce any significant alteration in the hemagglutinating activity of EiLL and EiSL, suggesting that arginine, histidine and cysteine residues, respectively, did not play any important role in the activity of either EiSL or EiLL. Amino acid analysis of these two proteins, further supported the fact that indicated an absence of cys-

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teine. However, total loss of both lectin activities by NBS and DTNB was evident, whereas no change in the control was detected; these results strongly suggest involvement of tryptophan residues in lectin activities and stabilities. A 50% loss in activity of EiLL and EiSL upon treatment with NAI suggested the partial necessity of tyrosine residue(s) in activities of EiLL and EiSL. A quantitative assay was done to determine the total number of tryptophan residues responsible for EiSL biological activity spectrophotometrically. Oxidation of the tryptophan indole moiety into oxindole by NBS is particularly interesting since the additional steric hindrance of the substituted group is very limited, contrary to other chemical modifiers [43]. EiSL was taken as a model for this study as it was not possible to perform the same work with EiLL due to a much smaller amount of lectin in leaves of Erythrina indica (≈ 600 µg/100 g of leaves), which is a hurdle for large scale characterization as well as practical implementations of vegetative tissue lectin [44]. Spectrophotometric monitoring revealed that the controlled addition of 10 µl of 10 mM NBS under mild acidic condition (pH 6.0) led to a progressive decrease in absorption at 280 nm indicative of oxidation of tryptophan residue. The absence of an increase in the absorbance at 280 nm upon addition of NBS indicated the absence of unspecific modification of tyrosine or phenylalanine residues [45]. Restriction of the experimental conditions to acidic pH has the advantages of selective modification of the tryptophan residue [43], as well as rapid reactivity [27]. A 50% drop in EiSL activity was clear upon modification of a single tryptophan residue, whereas almost total loss of activity was observed upon modification of the second tryptophan residue (Fig. 5 ). 3.9. Fluorescence emission measurements A decrease in the fluorescence emission was apparent when two tryptophan residues of EiSL were oxidized (Fig. 6 ). The observed fluorescence emission maxima at 330 nm are typical of tryptophan in protein, indicating that the fluorescence is solely due to tryptophan residues exposed on the protein surface [46]. The relatively small change in the spectra (≈ 18%) is attributed to the fact that only two tryptophan residues have been modified out of the total reported number of tryptophan (13 residues) [See ref. 12]. There was no change in the emission maximum upon NBS modification, indicating that NBS-oxidation of tryptophan residues did not alter the environment of these residues [47]. To examine if the tryptophan residues modified by NBS are involved in the affinity site of EiSL, we have tried to protect the EiSL affinity site from inactivation by incubating lectin with 50 mM lactose for 30 min before addition of NBS. The inactivation of EiSL by NBS proceeded in the presence of lactose. This result indicated that although a tryptophan residue is necessary for a fully active EiSL, it is probably not directly involved in the ligand binding site of lectin, a similar result being obtained by Wink et al. with ecto-apyrase enzyme from

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4. Conclusion Despite the environmental and geographical variations in Erythrina species distribution, their lectins share apparent similarities in their biological and molecular characteristics. This high degree of resemblance in their physicochemical parameters may suggest that Erythrina lectins are assigned to perform an important biological role(s) inside the plant microenvironment. Modification of EiSL with affinity chemical reagents indicated the importance of tyrosine and tryptophan in maintaining EiSL activity and stability. The tryptophan residue seems to play a structural role rather than binding haptenic sugar.

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[4] Fig. 5. Titration of EiSL with NBS. Tryptophan residues were quantified by stepwise addition of NBS as described in the text.

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Fig. 6. Fluorescence spectra of the native and modified EiSL. EiSL (100 µg/ml) was excited at 295 nm, and spectra were recorded between 300 and 400 nm.

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