Isolation of lectin and albumin from Pisum sativum var. macrocarpon ser. cv. sugar snap

The International Journal of Biochemistry & Cell Biology 33 (2001) 95–102 www.elsevier.com/locate/ijbcb

Isolation of lectin and albumin from Pisum sati6um var. macrocarpon ser. cv. sugar snap Xiuyun Ye, T.B. Ng * Department of Biochemistry, Faculty of Medicine, The Chinese Uni6ersity of Hong Kong, Shatin, Hong Kong Received 18 April 2000; received in revised form 18 July 2000; accepted 20 July 2000

Abstract A mannose- and glucose-binding lectin bearing considerable sequence similarity to other legume lectins was isolated using a simple procedure, from legumes of the sugar snap Pisum sati6um var. macrocarpon. The lectin was unadsorbed on Affi-gel blue gel and Q-Sepharose in 10 mM Tris – HCl buffer (pH 7.2) and adsorbed on SP-Toyopearl in 50 mM NaOAc buffer (pH 5). An albumin could also be purified at the same time. It was unadsorbed on Affi-gel Blue gel, adsorbed on Q-Sepharose and unadsorbed on SP-Toyopearl under the aforementioned chromatographic conditions. The lectin was almost identical in N-terminal sequences of its a- and b-subunit to lectin from P. sati6um L. var. Feltham First except for the 19th N-terminal residue of the b-subunit. The lectin was devoid of antifungal activity. Out of the 15 N-terminal amino acids examined in pea albumin, three were different between the two varieties of P. sati6um. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Pisum sati6um; Lectin; Albumin

1. Introduction A lectin could be prepared from Pisum sati6um L. by chromatography on Sephadex G100. The lectin is a nonglycosylated tetramer (a2b2) with a molecular weight of about 50 kDa, 17 kDa for b-subunit and 6 kDa for a-subunit. It is specific for mannose and glucose [1 – 3]. The gene sequence of pea seed lectin was reported by Gatehouse et al. [4]. Hoedemaeker et al. [5] gave an account of the complete sequences of * Corresponding author. Tel.: +852-26096875; fax: + 85226035123. E-mail address: [email protected] (T.B. Ng).

the a-subunits of pea seed and root lectin, the C-terminal amino acids of the b-subunits of pea seed lectin and most of the sequence of the b-subunits of pea root lectin. Pea root lectin is identical to pea seed isolectin 2. The crystal structure of the pea lectin has been determined by Einspahr et al. [6]. The design, expression and crystallization of recombinant pea lectin have been reported by Prasthofer et al. [7]. The conformational stability of the homodimeric pea lectin has been determined by both isothermal urea-induced and thermal denaturation in the absence and presence of urea. The relatively high conformational stability may reflect a relatively large size of the dimeric molecule (MW 49 kDa) and probably a conse-

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quent larger buried hydrophobic core in the folded protein. The simple two-state nature of the unfolding process with the absence of any monomeric intermediate suggests significant contribution of quaternary interactions to conformational stability of the oligomer [8]. A simple procedure is described herein for the purification of lectin from legumes (pods plus seeds) of the sugar snap, P. sati6um var. macrocarpon ser. cv. sugar snap. The lectin shows a replacement of W, the 19th residue in the b-chain, by E when compared with P. sati6um L. var. Feltham First. The procedure is also efficient for isolating albumin from pea legumes. Pea albumin is a sulfur-rich protein with a low molecular weight [9,10]. The level of mRNA for pea albumin is reduced in sulfur deficiency and downstream elements from the pea albumin 1 gene confer sulfur responsiveness on a reporter gene [11].

2. Materials and methods Fresh sugar snap (honey pea) legumes were obtained from a local market. They were authenticated to be P. sati6um var. macrocarpon ser. cv sugar snap by Professor S.Y. Hu at the Herbarium of the Chinese University of Hong Kong. The legumes were homogenized in distilled water (0.3 g/ml). The homogenate was then centrifuged 10 000× g, 20 min and the supernatant was dialyzed overnight against distilled water. Tris – HCl buffer (pH 7.2) was added to the dialyzed supernatant so that the final concentration of Tris was 10 mM before application to a column of Affi-gel Blue gel (2.5× 18 cm). The unadsorbed proteins were removed by washing the column with 10 mM Tris –HCl buffer (pH 7.2) and then directly chromatographed on a column of Q-Sepharose (1.5 × 18 cm). Unadsorbed proteins were removed by eluting with 10 mM Tris – HCl buffer (pH 7.2) and adsorbed proteins were removed by washing the column with a NaCl gradient (0 – 0.5 M). The unadsorbed proteins were dialyzed, lyophilized, dissolved in 50 mM NaOAc buffer (pH 5) and applied on a column of SP-Toyopearl 650M column (1.5 ×18 cm) which had been equilibrated and eluted with the same buffer. After unadsorbed

proteins had been eluted, adsorbed proteins were desorbed with a linear NaCl gradient (0–500 mM) in the same buffer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli and Favre [12]. N-terminal sequencing of the purified protein was conducted using a Hewlett-Packard G-1000A Edman degradition unit and an HP 1000 HPLC system. The assay for hemagglutinating activity was conducted in microtiter plates. A solution of the lectin (50 ml), serially diluted 2-fold, was mixed with a 2% suspension of rabbit erythrocytes (50 ml) in phosphate-buffered saline (0.2 M, pH 7.2). The results were recorded approximately 1 h later when the blank had fully sedimented. The hemagglutination titer is defined as the reciprocal of the highest dilution exhibiting hemagglutination and is equivalent to one hemagglutination unit. Specific hemagglutinating activity is expressed as the number of hemagglutinating units per mg protein [13]. Test of inhibition of lectin-induced hemagglutination by various carbohydrates was carried out as follows. A solution of lectin with 4 hemagglutination units was mixed with an equal volume of a serial 2-fold dilution of the carbohydrate sample to be tested. After incubation at room temperature for 30 min, the mixture was mixed with a 2% suspension of rabbit erythrocytes. The minimal concentration of the carbohydrate in the final reaction mixture capable of completely inhibiting 4 hemagglutination units of the lectin was calculated from the results [13]. The assay for antifungal activity was performed using sterile petri plates (100×15 mm) containing 10 ml potato dextrose agar [14]. Prior to the assay the fungi had been grown on the plate for 3 days. At the center of the plate was placed a sterile blank paper disk (0.625 cm in diameter). Around this central disk and at a distance of 1 cm from it were placed paper disks of the same size. A 6-ml aliquot of the lectin containing 300 or 60 mg in 10 mM sodium acetate buffer (pH 5.5) containing 0.13 M NaCl was added to a disk. Incubation of the petri plate was carried out at 23°C for 72 h until mycelial growth from the central disk had enveloped peripheral disks containing the control and had formed crescents of inhibition around disks with

X. Ye, T.B. Ng / The International Journal of Biochemistry & Cell Biology 33 (2001) 95–102

Fig. 1. Fractionation of the crude extract of sugar snap legumes (1215 mg) on an Affi-Gel Blue Gel column (2.5 × 18 cm) equilibrated with the binding buffer 10 mM Tris–HCl, pH 7.2. The gel was washed with binding buffer and eluted with a linear gradient of 0 – 500 mM NaCl in the same buffer.

antifungal samples. Four fungal species, Fusarium oxysporum, Rhizoctonia solani, Botrytis cinerea and Mycosphaerella arachidicola were examined in the assay. The positive control used was antifungal protein (thaumatin-like protein) from French beans [14]. The negative control were buffer and bovine serum albumin.

3. Results Chromatography of the legume extract on Affigel Blue gel yielded a large unadsorbed fraction (fraction A) and a smaller adsorbed fraction (Fig. 1). Lectin activity resided in the former fraction (Table 1) which was then fractionated on Q-Sepharose into an unadsorbed fraction (B) and four adsorbed peaks (Fig. 2). Lectin activity was detected again in the unadsorbed fraction (Table 1).

Fig. 2. Chromatography of fraction A (832.5 mg) of lectin activity from Affi-Gel Blue Gel on Q-Sepharose column (1.5 × 18 cm). The sample was applied on the column directly. After unadsorbed proteins (reaction B) had come off the column, adsorbed proteins were eluted with a linear gradient from 0 to 500 mM NaCl in the Tris – HCl buffer (10 mM, pH 7.2).

When the unadsorbed fraction was chromatographed on SP-Toyopearl 650M, only a very small portion was bound to the gel. This bound fraction contained purified lectin (Fig. 3). The lectin was dissociated into its a- and b-subunits in SDS-PAGE with a molecular weight of 17 and 6 kDa, respectively (Fig. 4). The protein yields and specific hemagglutinating activities of the various lectin-enriched fractions at different stages of the purification process are shown in Table 1. The N-terminal amino acid sequences of the a- and b-subunits of the purified lectin are presented in Tables 2 and 3. The sequence of a-subunit is identical to that reported in the literature. The sequence of the b-subunit is identical except for the substitution of W at position 19 by E of the various legume lectins examined, replacement of this W residue occurs only in a few species (Table 2). It is substituted by F in Glycyrrhiza glabra and

Table 1 Recovery of protein and hemagglutinating activity during purification of lectin from sugar snap legumes (150 g) Fraction

Protein (mg)

Crude extract 1215 Affi-Gel Blue Gel (Fraction 832.5 A) Q-Sepharose (Fraction B) 338.4 SP-Toyopearl 2.8

97

Specific hemagglutinating activity (U/mg)

Total hemagglutinating activity (U)

474.1 691.9

576 031 576 007

756.5 41 795.9

256 000 117 029

1 1 1 1 1 2 184 2 183 2 216 212 83 74 185 209 203 223 207 221 196 186 83

Pisum sati6um (this study) Arachis hypogaea-aa Bird 6etch Lathyrus hirsutus-ab Lathyrus tingitanus-a Lathyrus aphaca-a Lathyrus missolia Spring 6etch-a Fa6a bean Lathyrus ochrus c Medicago truncatula Medicago sati6a Glycyrrhiza glabra Vicia cracca Psophocarpus tetragonolobus Phaseolus acutifolius Sophora japonica Cladrastis lutea Phaseolus 6ulgaris Robinia pseudoacacia Cystisus scoparius d Lima bean Galega orientalis

V6 T6 S6 Y6 T6 L6 S6 D6 V6 V6 S6 L6 K6 D6 V6 V6 P6 E6 E6 V6 R6 I6 G6 F6 S6 A6 T6 T6 G6 A6 W6 P6 W6 NE P6 W6 NEI P6 W6 NE A6 EF W6 NE P6 I6 W6 V6 G6 A6 P6 I6 W6 V6 L6 G6 E6 P6 W6 NE P6 W6 I6 P6 W6 A6 I6 P6 W6 A6 S6 IV S6 D6 L6 FA P6 W6 I6 IA I6 D6 Q6 L6 S6 VV A6 FIV T6 D6 S6 L6 W6 V6 I6 AA D6 SIL W6 V6 T6 I6 AA D6 SIL W6 V6 SA FIV T6 D6 S6 L6 W6 SV IV S6 D6 Q6 L6 QF DV II AN D6 AT W6 II EK S6 W6 N6 N6 L6 W6 30 30 30 30 30 30 211 31 212 30 243 239 110 96 212 236 230 253 234 248 223 213 103

Residue no.

100 96 90 86 83 82 82 86 83 79 82 82 75 91 60 64 64 61 64 64 71 64 85

% Identity

b

Also including Pisum sati6um lectin sequence reported in literature. Also including Lathyrus odoratus-a, Lathyrus cicera-a, chickling vetch-a, lentil lectin-D. c Also including Lathyrus clymenum lectin. d Also including scotch broom lectin. e Only residues different from the corresponding residues in the sequence of Pisum sati6um lectin are shown for the other legume lectins. This residue is the only difference from the b-subunit sequence reported in the literature.

a

Residue no.

Lectin

Table 2 Comparison of N-terminal sequence of b-subunit of sugar snap (Pisum sati6um) lectin with other legume lectins (BLAST search results)e

98 X. Ye, T.B. Ng / The International Journal of Biochemistry & Cell Biology 33 (2001) 95–102

a

2 17 123 34

1 1 1 2 27 31 34 3 10 1

1

Residue no.

L F SV

QSL SDSL DALH V

QSL N D

I

G

SF D N ED TFNN G RD L MFNQ K KD L SWN V K P M L

A S P R P T Q K A IE Q N SF V EE L S P VSG P SF D N ED TFS K N P L

S S R

AHIPSGT AT AIV

NA SDG NATSNNV

D L G NAI L A SSSN Q

G

T ET TSFLITKFSPDQQNLIFQGDGYTTK E KLTL

Only residues different from the corresponding residues in the sequence of Pisum sati6um lectin are shown for the other legume lectins.

Leca dolla lectin Sophora japonica lectin Concanavalin A Glycine max lectin

Pisum sati6um lectin (this study) Lens culinaris lectin Lathyrus ochrus lectin Lathyrus nissolia lectin Vicia faba lectin Medicago sati6a lectin Medicago trunatcula lectin Robinia pseudoacacia lectin Lathyrus sphaericus lectin Dolichos lablab lectin Butea frondosa lectin

Lectin

Table 3 Comparison of N-terminal sequence of a-subunit of sugar snap (Pisum sati6um) with other lectinsa

22 49 148 59

33 33 33 33 53 63 56 43 39 23

33

Residue no.

57 39 44 53

93 90 90 81 74 60 65 51 46 60

100

% Identity

X. Ye, T.B. Ng / The International Journal of Biochemistry & Cell Biology 33 (2001) 95–102 99

100

X. Ye, T.B. Ng / The International Journal of Biochemistry & Cell Biology 33 (2001) 95–102

Robinia pseudoacacia lectins and by S in Psophocarpus tetragonolobus. The sequence similarities among the various lectins are also shown in Table 2. A comparison of the N-terminal sequences of the a-subunit of the various legume lectins is presented in Table 3. Generally speaking, those lectins with pronounced sequence similarity in a-subunit to P.

Table 4 Carbohydrate specificity of Sugar snap (Pisum sati6um) lectin Inhibitor (mM)

Minimum concentrationa

D(+)

0.98 NDb 7.81 ND 62.5 ND ND ND ND ND 1.95

mannose mannose D-mannosamine D(+)-galactose D(+)-glucosamine a-L(−)-fucose D(+)-galactosamine a-lactose a-D(+) melibiose a(+)-xylose D(+)-glucose L(−)

a Minimum concentrations required for inhibition of 4 hemagglutinating units of lectin. b ND, no inhibition of hemagglutination.

Fig. 3. Chromatography of fraction B (328.4 mg) of lectin activity from Q-Sepharose on SP-Toyopearl 650M column (1.5× 18 cm). Fractions with lectin activity pooled on Q-Sepharose were dialyzed against with deionized water and then lyophilized. The sample was dissolved in 50 mM acetate buffer (pH 5.0) and applied on a SP Toyopearl-650M column. After unadsorbed proteins had come off the column, adsorbed proteins were eluted with a linear gradient from 0 to 500 mM NaCl in the same buffer.

sati6um lectin also manifest marked sequence resemblance in b-subunits, and those lectins with a lower degree of sequence identity in a-subunit also possess a lesser extent of sequence identity in b-subunit. The P. sati6um lectin was devoid of antifungal activity against F. oxysporum, R. solani, B. cinerea and M. arachidicola while the red kidney bean lectin isolated in the laboratory possessed antifungal activity when tested at the same doses (300 and 60 mg) (data not shown). The carbohydrate specificity of the lectin was toward D-mannose and D-glucose. D-Mannosamine and D-glucosamine were less inhibitiory (Table 4). Albumin was present in the second adsorbed (P2) peak eluted from the Q-Sepharose column. It demonstrated a molecular weight of 11 kDa in SDS-PAGE. Its N-terminal sequence was only slightly different from that reported for another variety of P. sati6um (Table 5).

4. Discussion

Fig. 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS – PAGE, 15% gel) of sugar snap lectin. Left and middle lanes: sugar snap lectin (20 mg); right lane: Pharmacia molecular weight markers.

A relatively simple and efficient procedure is described herein for purifying lectin and albumin from legumes of a variety of P. sati6um different from the variety previously investigated [4,9]. The procedure entailed an initial step of adsorbing ribosome inactivating proteins and related proteins on the affinity chromatography medium

X. Ye, T.B. Ng / The International Journal of Biochemistry & Cell Biology 33 (2001) 95–102

Affi-gel Blue gel. This step has also been included in the isolation protocols of a number of ribosome inactivating proteins including a- and b-momorcharins from Momordica charantia seeds [15], new ribosome inactivating proteins from M. charantia fruits and seeds [16], and a- and b-pisavins from seeds of P. sati6um var. ar6ense Poir [17]. Proteins other than ribosome inactivating proteins and related proteins are unadsorbed. This is evidenced by the removal of a considerable amount of proteins in the elution profile of sugar snap extract from Affi-gel Blue gel and the approximately 50% increase in specific hemagglutinating activity. The next step of ion exchange chromatography on Q-Sepharose resulted in removal of a substantial quantity of adsorbed proteins although the specific hemagglutinating activity was not proportionately elevated. The final step of ion exchange chromatography on SP-Toyopearl was a powerful tool. A small lectin-containing peak was adsorbed and by comparison a much bigger peak devoid of lectin activity remained unadsorbed, resulting in a 5.5fold increase in specific hemagglutinating activity over the lectin-enriched fraction from the previous chromatographic step. As regards the pea albumin, the procedure utilized in the present investigation could also be used to separate it from other proteins. It was unadsorbed on Affi-gel Blue gel just like the lectin. However, its chromatographic behavior in subsequent steps including ion exchange chromatography on Q-Sepharose and SP-Toyopearl differed from that of lectin and could thus be easily separated from lectin. A DEAE-ion exchange column was utilized previously for purifying pea albumin which was adsorbed on the ion exchanger [9,10]. The procedure used by Entlicher et al. [1] for Table 5 Comparison of N-terminal sequence of sugar snap (Pisum sati6um var. macrocarpon) albumin with albumin from Pisum sati6um L. var. Feltham Firsta Pisum sati6um var. D macrocarpon A Pisum sati6um L. var. Feltham D First D6 a

E A E C6

H R H R

PNLLESDA K P N L C6 E S D A K

Only different residues are underlined.

101

purifying P. sati6um seed lectin involved extraction with water, (NH4)2SO4 precipitation, specific adsorption on Sephadex G-150 and ion exchange chromatography on DEAE-cellulose. The protocol employed by Gatehouse et al. [9] for isolating P. sati6um seed albumin entailed defatting with hexane, extraction with 20 mM NaOAc (pH 5), (NH4)2SO4 precipitation, chromatography on Sephadex G-75 and ion exchange chromatography on DEAE-cellulose. The procedures utilized in the present study did not involve (NH4)2SO4 precipitation and delipidation and were thus more convenient. The yields of the lectin from seeds was about 15 – 30 mg/150 g seeds [1], higher than the yield from legumes in the present study. It is noteworthy that a change in a single amino acid residue from W to E was detected in the b-subunits of the P. sati6um lectin isolated in the present study compared with the sequence available in the literature. When sequence comparison is made among the various legume lectins, it is observed that substitution of W by another residue is not a frequent occurrence. Out of the 25 lectins examined, replacement of W occurs in only three lectins. It appears that in general lectins whose b-subunits resemble sugar snap lectin more possess a-subunits exhibiting a greater extent of sequence identity to sugar snap lectin. The single amino acid substitution of W by E did not ensue in a change in the carbohydrate specificity of the P. sati6um lectin. Some lectins and lectin-like proteins may exhibit antifungal activity [18,19]. However, sugar snap lectin did not manifest activity toward the four fungal species examined. Whether lectin from other varieties of P. sati6um possesses antifungal activity has not been reported. The albumin isolated from the sugar snap had the same molecular weight but a slightly different N-terminal sequence from the previously reported pea albumin isolated from P. sati6um [9,10]. Higgins et al. [9] have conducted a sequence comparison of pea albumin with other low molecular weight seed albumins and protease inhibitors and detected only a low level of homology. Gatehouse et al. [9] suggested that pea albumin is one of the proteins synthesized by pea seeds in excess of their requirements.

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Lam et al. [17] demonstrated the existence of two ribosome inactivating proteins, one with a molecular weight of 18 kDa and another with a molecular weight of 20 kDa although both with the same N-terminal sequence which resembles the sweet protein miraculin, from the seeds of P. sati6um var. ar6ense Poir. Ye et al. [20] isolated from sugar snap legumes an antifungal protein with low ribosome inactivating protein activity, a molecular weight of 36 kDa and a miraculin-like N-terminal sequence. These findings, together with the present observations, indicate that similar but not identical proteins are elaborated by different varieties of the same species. The extent of similarity may differ from protein to protein. Acknowledgements The expert secretarial assistance of Ms Fion Yung, Ms Grace Chan, Ms Iris Wong and Ms Janny Lee is appreciated. References [1] G. Entlicher, J.V. Kostir, J. Kocourek, Studies on phytohemagglutinins. III. Isolation and characterization of hemagglutinins from pea (Pisum sati6um L.), Biochim. Biophys. Acta 221 (1970) 272–281. [2] I.S. Trowbridge, Isolation and chemical characterization of a mitogenic lectin from Pisum sati6um, J. Biol. Chem. 249 (1974) 6004 – 6012. [3] E. Van Driessche, S. Vandenbranden, L. Kanarek, Improvement in the purification procedure of pea lectin, and considerations on the subunit structure, Arch. Int. Physiol. Biochim. 86 (1978) 963–964. [4] J.A. Gatehouse, D. Bown, I.M. Evans, I.N. Gatehouse, D. Jobes, P. Preston, R.R. Croy, Sequence of the seed lectin gene from pea (Pisum sati6um L.), Nucleic Acids Res. 15 (1987) 7642. [5] F.J. Hoedmaeker, M. Richardson, C.I. Diaz, B.S. de Pater, J.W. Kijne, Pea (Pisum sati6um L.) seed isolectins 1 and 2 and pea root lectin result from carboxypeptidase-like processing of a single gene product, Plant Mol. Biol. 24 (1994) 175 – 181. [6] H. Einspahr, E.H. Parks, K. Surguna, E. Subramanian, F.L. Suddarth, The crystal structure of pea lectin at 3.0-A resolution, J. Biol. Chem. 261 (1986) 16518–16527. [7] T. Prasthofer, S.R. Phillips, F.L. Suddarth, J.A. Ergler, Design, expression and crystallization of recombinant lectin from the garden pea (Pisum sati6um), J. Biol. Chem. 264 (1989) 6793 – 6796.

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