Novel mannose binding lectins from agricultural crops

Industrial Crops and Products 19 (2004) 137–146

Novel mannose binding lectins from agricultural crops

Elaine Davidson, Derek Stewart∗

Division of Plant Biochemistry and Cell Biology, Unit of Plant Biochemistry,Scottish Crop Research Institute, Dundee, DD2 5DA Scotland, UK

Abstract

As part of a large programme [Scottish Executive Rural Affairs Department Programme of Agricultural, Biological andRelated Research: Exploitation of Novel and Known Lectins in Agricultural and Biological Research—an InterdisciplinaryApproach to Improve Crop Protection and Productivity, Animal (Including Human) Welfare and Health (Project No. FF821).Scottish Executive Rural Affairs Department (http://www.scotland.gov.uk/abrg/docs/pabr-00.asp)] concerned with the discoveryand development of new and existing plant lectins, a screening exercise for novel mannose binding lectins (MBLs) was initiated.Common agricultural and horticultural crops were screened and of the 50 species initially screened, 15 were shown to beputative sources of MBLs. Following isolation by mannose-affinity and phenyl-SepharoseTM chromatographies the proteinswere characterised with regard to their structure and functionality. The species within theAmaryllidaceaandLiliaceaehadthe MBLs present at the greatest levels (≤6000�g [g fresh wt.]−1). The novel MBLs were present at relatively low levels(15–60�g [g fresh wt.]−1). All the MBLs exhibited monomer molecular weights in the range 11–13 kD whilst the native molecularweights were indicative of dimer or tetramer formation.

The linkage preferences of the MBLs were determined by inhibiting the MBL-yeast mannan precipitation reaction by theaddition of oligomannans with defined linkages. The preference for�(1–3) and�(1–6) linkages was predominant throughoutthe known and novel MBLs. Evidence of binding to�(1–2) and�(1–4) linked ligands was seen but the relative affinity for thesewas low with the exception of the MBL from leafbeet (Beta vulgarisssp. cicla). The MBL from celeriac (Apium graveolens)did bind�-linked ligands but this was most probably due to a lack of specificity rather than a different specificity. Overall therelative binding affinity of the MBLs increased with increasing complexity of the ligands.© 2003 Elsevier B.V. All rights reserved.

Keywords:Mannose-binding lectin; Plant; Specificity; Affinity

1. Introduction

The desire to derive materials and products via moreenvironmentally aware, recyclable processes is grow-ing apace (Sommerville and Bonetta, 2001; Mon et al.,

∗ Corresponding author. Tel.:+44-1382-568-517/562-731;fax: +44-1382-562-731.

E-mail address:[email protected] (D. Stewart).

1998; Fritz and Schroeter, 1994; Nessler, 1994;Frusciante et al., 2000). In particular, the desire to seeplants (crops) as multiple-use factories has becomea particular focus (Wilke, 1999) and indeed, can beseen as the raison d’etre of this journal.

Plant lectins have, over the past 20 years, been thesubject of much research regarding their evolution(Van Damme et al., 1995, 1998a), biosynthesis (VanDamme et al., 1991a,b), function (Chrispeels and

0926-6690/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.indcrop.2003.08.001

http://www.scotland.gov.uk/abrg/docs/pabr-00.asp

138 E. Davidson, D. Stewart / Industrial Crops and Products 19 (2004) 137–146

Raikhel, 1991; Peumans and Van Damme, 1995a,b;Rüdiger, 1998) and application in many diversefields such as plant protection (Birch et al., 1999;Hilder et al., 1995), glycoprotein isolation (Debrayand Montreuil, 1991; Shibuya et al., 1988) and, per-haps more importantly, the diagnosis and treatment ofmammalian diseases (Schumacher, 1995; Remmelinket al., 1999). Indeed it is in this latter area of re-search and in particular the field of immunode-ficiency virus-related research that the monocot,mannose-specific lectins (MSLs) have come toprominence (Weiler et al., 1990; Stewart et al.,1991).

Van Damme et al. (1998b)have shown that themonocot MSLs exhibit a very precise specificity formannose and, in addition, a further preference fordistinct glycosidic linkages. For example, the lectinsfrom Galanthus nivalus(GNA, snowdrop) andNarcis-sus pseudonarcissus(NPA, daffodil) display definitepreferences for�(1–3) and�(1–6) mannose–mannoselinkages (Fig. 1), respectively. Although other exam-ples of MSLs have been reported for otherAmaryl-

HO

OHO

O

OHO

OH

O

O

OH

HO

O

HOOH

HOH2C

O

OHOHO

HOH2COH

OHOHO

HOH2COH

α(1-6)

α(1-3)

Fig. 1. Chemical representation of a mannopentose. The�(1–3) and�(1–6) highlighted and the�(1–2) bonds are represented by - - -.

lidaceae(Van Damme et al., 1988; Peumans et al.,1991) and other, generally ornamental (Van Dammeet al., 1994), plant families, the allium-derived lectinsincluding the onion, shallot and ramson (Smeetset al., 1997; Van Damme et al., 1993), are the onlyones classed as crops. However, the issue becomesclouded when the topic of specificity is addressedsince there is another group of lectins reported tobind mannose but with a reduced specificity; themannose-binding jacalin-related lectins. Jacalin is alectin from the seeds of the jackfruit,Artocarpus in-tegrifolia, a member of theMoraceaefamily whichexhibit a preference for GalNAc. However, there is asub group of lectins which are sequence, structurallyand evolutionary-related to jacalin and these comprisemembers of many taxonomically unrelated familiessuch asMusaceae(banana),Asteraceae(Jerusalemartichoke), Graminaea (barley, rice), Brassicaceae(oilseed rape) andConvolvulaceaea (hedgebindweed), which preferentially bind mannose/maltose(Geshi and Brandy, 1998; Hirano et al., 2000; Peumanset al., 2000; Houles Astoul et al., 2002).

E. Davidson, D. Stewart / Industrial Crops and Products 19 (2004) 137–146 139

Our aim was to look at established crops and todetermine whether they contained lectins that specif-ically bound mannose and, if so, to isolate and char-acterise them (Anon, 2000). We report the results ofthese studies here.

2. Materials and methods

All plant material was either purchased locally orobtained from Seed-By-Size Ltd (Hemel Hempstead,UK). Mannosidases were from CN Biosciences Ltd,UK. Chemicals and chromatographic media werefrom Merck and Sigma–Aldrich, UK, unless statedotherwise.�(1–2), �(1–3) and�(1–6) Mannobiosesand Man�(1–3)[Man�(1–6)]Man were purchasedfrom Dextra Laboratories (UK). Man�(1–6)Glc andMan�(1–2)[Man�(1–4)]Man were gifts from Dr.L.C.N. Tucker, University of Dundee.� and�-methylmannosides were synthesised as described byStewart(1993). Man�(1–6)Man�(1–6)Man, and�(1–4) man-nobiose and mannotriose, were isolated from a limitedtrifluroacetic acid (TFA) hydrolysis ofS. cerevisiaeand ivory nut mannans, respectively.

The complex oligomannosides Man′5-(GlcNAc)2

Asn, Man′′5-(GlcNAc)2Asn and Man6-(GlcNAc)2Asnwere derived from porcine thyroglobulin (Sigma–Aldrich, UK) using the hydrazinolysis method ofTakasaki et al. (1982). The oligomannosides wereseparated using RP-HPLC and a semi-prep C8 col-umn (250 mm× 25 mm, Lichrom, Merck, UK).The oligomannosides dissolved in 0.5% TFA indH2O (10 mg ml−1) and eluted from the column(1 ml min−1) using a gradient of 0.05 M TFA in dH2Oto 0.05 M TFA in 80% methanol. Fractions (0.5 ml)were collected and assayed for polysaccharide usingthe phenol sulphuric acid method ofDubois et al.(1956)

2.1. Lectin purification

The extraction procedure followed was that de-scribed byVan Damme et al. (1988). Briefly, plantmaterial was rapidly chopped into small pieces and ho-mogenised in 1 M (NH4)2SO4 (10 ml [g fresh wt.]−1).The homogenate was then centrifuged (25,000× g

for 30 min) and the supernatant frozen overnightat −80◦C. Once thawed the homogenate was

re-centrifuged as before to remove any precipitate. Theclear supernatant was applied to a mannose-agarosecolumn (1.5 cm × 20 cm) pre-equilibrated with1 M (NH4)2SO4. Three column volumes of 1 M(NH4)2SO4 were run through to remove unboundmaterial. Bound (lectin) material was eluted with20 mM 2,3-diaminopropane. Eluate was monitored at280 nm. The eluate was concentrated to 5 ml by rotaryevaporation (<40◦C), made up to 1 M (NH4)2SO4and the concentrate applied to a phenyl-SepharoseTM

column (1.5 cm × 20 cm) pre-equilibrated with 1 M(NH4)2SO4. Two column volumes of 1 M (NH4)2SO4were run through the column to remove phenolicsand the lectin desorbed by elution with dH2O. Eluatewas monitored at 280 nm and tested for protein usingthe using the BioRad Protein Assay (BioRad, UK).The combined protein eluate was freeze-dried.

2.2. Mannosidase activity

� and� mannosidase activity was assessed as de-scribed byMcCleary (1988). The Calibration curveswere constructed using a carob-galactomannan/�(1–2,3, 6) mannosidase (ex. Jack Bean) and�(1–4) woodmannan/�(1–4) mannosidase (exXanthomonas holci-cola).

2.3. Haemagglutination assay

Haemagglutination assays were carried out using96-well U bottomed microplates (Corning Costar)with rabbit erythrocytes (Scottish Antibody Produc-tion Unit, UK) washed and diluted in phosphatebuffered saline (PBS) at a ratio of 1:10. Freeze-driedlectins were reconstituted at 2 mg/ml and 50�l addedto a well and in serially diluted with 100�l of PBSacross the plate. Blood (20�l) was then added toeach well and incubated at room temperature (20◦C)for approximately 3 h. Agglutination was detectedvisually.

2.4. Sodium dodecyl sulphate-polyacrylamide gelelectrophoresis

The lectin monomer molecular weight determina-tions were done using 15.0% (w/v) acrylamide gelsunder reducing conditions essentially as described byLaemmli (1970).


2.5. Lectin molecular weight determination

Native molecular weights were determined by gelfiltration chromatography on a Gilson HPLC usinga Zorbax, GF-250 4.6 mm × 250 mm column (Agi-lent Technologies, UK) and eluted at 1 ml min−1 with0.2 M Na2HPO4, pH 7.0. Eluate was monitored at280 nm.

2.6. Quantitative precipitation andprecipitation-inhibition assays

Precipitation reactions were conducted followingthe method ofSo and Goldstein (1967). Varyingamounts of the soluble mannan fromS. cerevisiae(0–80�g) were incubated with 30�g of MBL in a100�l of 10 mM phosphate buffer, pH 7.4, containing0.1 M CaCl2, 0.04% NaN3 and 150 mM NaCl. Afterincubation at 37◦C for 60 min, the mixtures were keptat 4◦C for 2 days and then centrifuged at 8800× g

for 30 min. Protein in the precipitates was determinedusing the BioRad Protein Assay (BioRad, UK) cali-brated with bovine serum albumin. This allowed theoptimum amounts of bothS. cerevisiaemannan andMBL necessary for maximum precipitation to be cal-culated. Experiments with all of the MBLs exhibitedvery similar precipitation curves (data not shown).This similarity meant that all the oligosaccharideprecipitation-inhibition assays were conducted using30�g of MBL and 20�g of S. cerevisiaemannan.These were incubated at 37◦C in 100�l of 10 mMphosphate buffer, pH 7.4, containing 0.1 M CaCl2,0.04% NaN3 and 150 mM NaCl. Increasing amountsof mono/oligomannosides were added to the solutioncontaining the mannan and MBL. The concentrationof mono/oligomannosides (mM) required to obtain a50% inhibition of precipitation was determined anda relative inhibitory potency (Rel-I-P) ranking wasestablished withd-mannose ranked at 1.

3. Results and discussion

3.1. Structure

Analysis of all the putative mannose bindinglectins (MBLs) by reducing SDS-PAGE showed thatthey all had monomeric molecular weights in the

range 11.0–13 kD (Table 1). This is certainly withinthe range for the monocot mannose-specific lectins(GNA-HHA; Van Damme et al., 1991a,b, 1998a,b)and slightly greater than that reported for the man-nose binding jacalin-related lectins; 14–15 kD (Hiranoet al., 2000; Peumans et al., 2000).

The MBLs displayed distinct differences with re-gard to their native molecular weights with evidencefrom gel filtration studies suggesting that, in vivo, thelectins exist either as dimers or tetramers (Table 1).There was no pattern for the appearance of the nativelectin as dimer or tetramer either within family orspecies. For example, of the fourAllium (and Lili-aceae) derived MBLs, three exist as dimers whilstthe remaining one (ACA,Table 1) is tetrameric.Conversely, two of theAmaryllidaceaeMBLs weretetrameric whilst the other (NPA) was dimeric. BoththeChenopodiaceae, Brassicaceaewere all tetramericwhilst theAsteraceaewere dimeric.

3.2. Lectin ligand binding

Relative mannose linkage binding affinity was de-termined using precipitation-inhibition assays employ-ing the highly branched mannan fromS. cerevisiae.Inthis assay quantitative precipitation curves were estab-lished for each MBL. Selected oligomannans, with de-fined mannose-mannose linkages were used to inhibitthe precipitation of the MBL/mannan system. The con-centration of oligomannan (mM) required to obtain a50% inhibition of precipitation was determined anda relative inhibitory potency ranking was establishedwith �-d-mannose ranked at 1. The results of these ex-periments are shown inTable 1. (The concentrationsrequired for 50% inhibition withd-mannose are inparenthesis). None of the MBLs exhibited exo and/orendo mannosidase activity (data not shown).

Even with the simplest glycoside, methyl manno-side, there were differences between the MBL ligandaffinities. With the exception of the celeriac MBL(AGA), they all preferentially bound to�-methylmannoside, although the difference for some, suchas BOGA, was marginal. However, some did dis-play a significant preference for the� linkage, suchas GNA, NPA and ACA. Progression to more com-plex �-linked mannobioses and mannotriose virtuallyeliminated inhibition of lectin binding ability exceptfor AGA. In fact AGA displayed significant binding

E.

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rop

sa

nd

Pro

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6141

Table 1The mannose binding preferences of mannose-specific lectins

Lectin Snowdrop (GNA) Daffodil (NPA) Shallot (AAA) Garlic (ASA) Ramson (AUA) Onion (ACA) Amaryllis (HHA) Beetroot (BVRA)

Monomer molecular weight (kD) 12.5 12.5 12.5 11.5/12.5 12.5 12.5 12.5 11.0Native molecular weight (kD) 50 25 25 25 25 50 50 42Yield (�g [g fresh wt.]−1) 3000 6500 300 250 1250 15 1100 40d-Man (IC 50 mM)) 1.0 (42) 1.0 (15) 1.0 (150) 1.0 (40) 1.0 (112) 1.0 (73) 1.0 (80) 1.0 (220)�-d-Man p-OMe 1.8 1.7 1.2 1.4 1.7 2.2 1.3 1.1�-d-Man p-OMe 0.1 0.3 0.6 0.4 0.3 0.4 0.7 0.2Man�(1–2)Man <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1Man�(1–2)Man�(1–2)Man <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1Man�(1–4)Man <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1Man�(1–4)Man�(1–4)Man <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1Man�(1–2)Man 2.2 2.9 3.4 1.4 3.6 1.7 3.2 2.3Man�(1–3)Man 12.1 3.8 6.1 11.9 5.3 7.7 3.7 9.2Man�(1–4)Man 1.1 1.7 1.9 0.8 1.3 1.9 1.4 1.7Man�(1–6)Man 3.2 5.0 17.0 2.7 4.1 2.7 6.0 1.9Man�(1–3)Man�-OMe 11.7 4.8 10.9 12.1 10.2 11.4 10.5 12.2Man�(1–6)Man�-OMe 3.5 6.0 9.0 2.9 5.1 4.4 12.3 2.4Man�(1–6)Glc 2.3 2.0 1.9 0.7 4.2 3.9 4.4 1.5Man�(1–6)Man�(1–6)Man 2.5 6.7 9.0 5.6 7.0 2.4 22.7 2.4

2.1 0.9 2.0 2.7 2.2 3.0 4.4 4.2

1.9 2.3 2.2 1.1 2.4 3.1 1.7 1.3

22.1 5.2 17.0 12.9 11.3 12.0 14.2 19.8

28.0 30.1 27.1 34.0 30 28.8 33.2 26.7

30.1 28.3 21.8 22.0 35.0 22.6 47.3 18.1

Family Amaryllidaceae Amaryllidaceae Liliaceae Liliaceae Liliaceae Liliaceae Amaryllidaceae ChenopodiaceaeGenus and species Galanthus nivalus Narcissus

pseudonarcissusAlliumascalonicum

Alliumsativum

Alliumursinum

Alliumcepa

Hippeastrumhybrid

Beta vulgarisssp. rubra

142E

.D

avid

son

,D

.S

tewa

rt/Ind

ustria

lCro

ps

an

dP

rod

ucts

19

(20

04

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37

–1

46

Table 1 (Continued)

Erysimum (EPA) Chicory (CIA) Dahlia (DHA) Artichoke (CSA) Kohl (BOGA) Leafbeet (BVCA) Celeriac (AGA)

Monomer molecular weight (kD) 11.5 12.0 12.0 12.0 13.0 12.5 12.0Native molecular weight (kD) 46 24 24 24 52 50 24Yield (�g [g fresh wt.]−1) 20 35 10 55 45 50 10d-Man (IC 50 mM)) 1.0 (175) 1.0 (215) 1.0 (110) 1.0 (134) 1.0 (183) 1.0 (232) 1.0 (253)�-d-Man p-OMe 1.5 1.4 1.2 1.4 1.1 1.3 0.7�-d-Man p-OMe 0.4 0.5 0.2 0.5 0.9 0.7 1.5Man�(1–2)Man <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 1.7Man�(1–2)Man�(1–2)Man <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 2.1Man�(1–4)Man <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 3.7Man�(1–4)Man�(1–4)Man <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 5.6Man�(1–2)Man 1.9 3.3 1.2 2.1 1.9 5.2 2.2Man�(1–3)Man 2.3 5.6 4.2 11.6 6.7 4.2 2.1Man�(1–4)Man 1.4 1.0 2.3 1.5 2.2 3.7 1.1Man�(1–6)Man 6.7 3.4 10.3 3.1 5.2 2.1 1.7Man�(1–3)Man�-OMe 4.2 6.5 6.7 18.7 4.3 5.8 3.4Man�(1–6)Man�-OMe 7.7 5.5 14.0 5.2 5.7 3.1 1.9Man�(1–6)Glc 3.4 1.9 5.7 2.4 3.2 3.3 2.1Man�(1–6)Man�(1–6)Man 7.6 5.5 12.3 2.9 4.9 4.1 3.7

5.1 5.2 5.2 5.6 11.7 15.3 2.7

2.2 2.1 3.2 2.2 6.7 13.7 3.4

9.0 14.5 12.2 14.7 5.3 9.7 2.7

22.3 24.7 19.5 25.7 8.4 6.0 4.4

23.1 23.8 22.7 14.3 15.4 15.4 5.4

Family Cruciferae Compositae Compositae Compositae Cruciferae Chenopodiaceae UmbelliferaeGenus and species Erysimum perofskianum Cichorium intybus Dahlia hoertensis Cynara scolymus Brassica oleracea B. vulgarisssp.cicla Apium graveolens

The concentration of oligomannan (mM) required to obtain a 50% inhibition of the MSL-S. cerevisiaeprecipitation reaction was determined and a relative inhibitory potency ranking wasestablished withd-mannose ranked at 1. The concentration of mannose (mM) required to obtain a 50% inhibition is in brackets.


to the�-linked ligands with a preference for those con-taining a�(1–4) linkage and, in particular, the morecomplex�(1–4) mannotriose.

It was with the progression to the more complex�-linked ligands that differences between the MBLsbecame apparent. Incubation with�(1–2) manno-biose generally resulted in increased Rel-I-P valueswith BCVA exhibiting the optimal increase (1.3–5.2).However, the values for some MBLs remained un-changed (ASA, DHA) or actually decreased (ACA;c.f. �-linked ligands). The changes in Rel-I-P valueswere even less significant following incubation with�(1–4) mannobiose. As before, BCVA experiencedthe largest change (1.3–3.7). The other member ofthe Chenopodiaceaefamily, Beetroot (BVRA) hadsmaller absolute Rel-I-P values for�(1–2) and (1–4)mannobiosides (2.3 and 1.7, respectively) but theirrelative ratio was the same as that for BVCA; at∼1.4(BVCA, 5.2/3.7 = 1.4; BVRA, 2.3/1.7 = 1.3).

Combining both the�(1–2) and�(1–4) linkages inone ligand, covalently bonded to�-methyl mannoside,did not significantly alter the Rel-I-P values. In factthe general trend was for a decrease but, as before,BVCA was exceptional, producing a 2.6 and 3.7 foldincrease in the Rel-I-P value relative to those obtainedfor �(1–2) and�(1–4) mannobiose, respectively, togive a value of 13.7.

More significant changes in the Rel-I-P values ac-companied incubation with ligands containing�(1–3)and�(1–6) linkages. The corresponding mannobiosesand�-methyl mannobiosides all increased the Rel-I-Pvalues (relative to�-methyl mannose) and to a greaterdegree than was seen following incubation with�(1–2)and�(1–4) mannobiose.

This specificity behaviour may give some clue as tothe type of lectins isolated from theBrassicaceae(EPAand BOGA) andAsteraceae(CIA, DHA, and CSA).Both of these families have previously been reported tocontain plants which have jacalin-like mannose bind-ing proteins (Bourne et al., 1999; Houles Astoul et al.,2002). Ligand specificity studies on some jacalin-likemannose binding lectins (derived from Jerusalem arti-choke; Asteraceae family) showed that they exhibiteda specificity towards�(1–2) and�(1–3) mannosidiclinkages. Here the binding to these linkages is evidentbut with the preference more towards�(1–3).

The �(1–3) linked mannobiose and methyl man-nobioside produced a relative increase in the overall

Rel-I-P values of 2.8–13.3-fold. The corresponding�(1–6) linked ligands produced a 1.2–14.1 relative in-crease.

Not all the elevated Rel-I-P values rested with theknown MBLs. For example, both BVRA and CSA ex-hibited significantly high Rel-I-P vales for the�(1–3)mannobiose (9.2 and 11.6, respectively) and thesewere further elevated upon methylation of the reduc-ing terminus (12.2 and 18.7, respectively). Interest-ingly, the values for the corresponding�(1–6) linkedligands were much lower giving a clear indication thatthese MBLs preferentially bind ligands containing aninternal (or non-terminal)�(1–3) mannose linkage.

Conversely, the Rel-I-P values suggest that a dis-tinct preference for an�(1–6) intra-mannose linkagewas evident for AAA, HHA and, to a lesser extent,DHA and EPA which is in broad agreement with thefindings of Kaku et al. (1992). However, for AAAmethylation of the reducing terminus resulted in a re-duction in the Rel-I-P value from 17.0 to 9.0. The cor-responding change for DHA was from 10.3 to 14.0.This is an indicator that AAA and DHA exhibit prefer-ences for reducing terminal and non-reducing terminal(internal) mannose linkages, respectively. The prefer-ence for an�(1–6) intra-mannose linkage was furthersupported following incubation with the�(1–6) man-notriose which resulted in a relatively large Rel-I-Pvalue. In particular HHA gave a high Rel-I-P value at22.7 with this ligand compared with that obtained forthat obtained with�(1–6) methyl mannobiose (12.3).Interestingly, HHA also exhibited relatively large val-ues for�(1–3) methyl mannobioside (10.5) but not thecorresponding free mannobiose (3.7). This may sug-gest that this lectin favours internal mannose linkagesand that the specificity is not exclusive, but favouredtowards,�(1–6).

Inhibition with a methyl mannotrioside containinga combined�(1–3)/�(1–6) linkage produced mixedresults in comparison to the corresponding (methy-lated) mannobioses. Only GNA, BVRA, CEA andBVCA exhibited significant increases in Rel-I-P. Sur-prisingly many of the MBLs had reduced Rel-I-P val-ues although these reductions were relatively small, ofthe order of 15–25% compared with the (methylated)mannobioses.

The addition of�(1–2) linked mannose residues toboth the non-reducing termini of the�(1–3)/�(1–6)residues resulted in a reduction in the Rel-I-P values


(c.f. the�(1–3)/�(1–6) methyl mannotrioside) for allthe MBLs with the exception of BOGA and BVCA.Interestingly, the use of the dual�(1–2) mannosylatedpentasaccharide increased Rel-I-P values for theseMBLs by 120 and 57%, respectively. However, inhi-bition of the NPA precipitation with this ligand pro-duced a very low Rel-I-P value of 0.9. The reason forthe general reduction may be steric hindrance (Fig. 1).Rotation about the�(1–2) linkage is like to impedeaccess to the�(1–3) and �(1–6) linkages therebyreducing the inhibitory power of this oligosaccharidewhen incubated with an MBL favouring�(1–3) and�(1–6) linkages.

The use of the other pentamannoside and the sexa-mannoside in the inhibition assay resulted in signifi-cant increases in the Rel-I-P values for all but BOGA,BVCA and AGA. The increases ranged from 25%(BVRA, c.f. �(1–3) methyl mannobiose) to 349%(NPA, c.f.�(1–6)mannotrioside). The absolute, ratherthan relative, size of the Rel-I-P values reflects theaffinity that the MBLs have, in general, for morecomplex oligo- and polysaccharides. In particularNPA, ASA, AUA and HHA all had Rel-I-P valuesat, or greater than, 30 reflecting their affinity for thecomplex ligands.

Further mannosylation of the pentamannoside withan �(1–2) mannose residue produced mixed results.Although some of the MBLs experienced reductionsin Rel-I-P they were not to the extent seen follow-ing the addition of two�(1–2) mannose residues tothe�(1–3)/�(1–6) mannotrioside. For example AAA,ASA, ACA, BVRA and CSA all experienced largerelative reductions in the inhibitory potential. Thelarge reduction in the Rel-I-P value for CSA coupledwith the large value obtained when�(1–3) methyl-mannoside was used for inhibition (18.7) suggeststhat this MLS displays a preference for�(1–3) link-ages that are not at the reducing terminus but at theother end of the polysaccharide; the non-reducingterminus.

Surprisingly GNA, AUA and in particular, HHAall experienced increased inhibition with the sex-asaccharide. HHA was shown earlier to exhibit apreference for�(1–6) linkages but whilst still bind-ing to �(1–3) ones. Possibly the masking of oneof the �(1–3) linkages by steric hindrance (due tothe �(1–2) linkage) has prompted greater�(1–6)binding.

4. Conclusion

These preliminary investigations have shown thatmannose binding lectins, be they “specific” or otherwise, are widespread throughout agriculturally im-portant crops. Since the proteins extracted, using astandard extraction protocol for MBLs, were shown todisplay native and monomer (lectin subunit) molecularweights similar to those previously reported for mem-bers of the MBL superfamily the inclination wouldbe to suggest a possible relationship. However, thejacalin-related lectins also have monomeric molecularweights at only a slightly greater value (14–15 kD).Definitive statements regarding the relatedness ofthese novel lectins to either the monocot-derivedand/or jacalin-related lectins cannot be made withoutrecourse to detailed biochemical and molecular analy-sis. Such analyses are outwith the scope of this paperand will form the basis of further research. Howeversome indication regarding the nature of the lectinsfrom the Brassicaceaea and Asteraceaea families weregleaned from the binding preferences which give apreliminary suggestion that they may be jacalin-like.Nevertheless, and perhaps more importantly, the ac-tual binding specificities and preferences have beencharacterised in some detail and it is these which willform the basis of any subsequent lectin use.

The linkage specificities were, in the majority,towards �(1–3) and�(1–6) linkages. However, theleafbeet MBL (BVCA) did preferentially bind�(1–2)linkages but this may be an indicator of a lack ofspecificity for position of linkage the second residuein an�(1-R) linked mannose–mannose linkage.

The novel MBLs, like the known (mainly MSL)ones, targeted�(1–3) and�(1–6) linkages and al-though low in some instances (BOGA and AGA) theyshowed affinities for the ligands at levels approachingthose previously reported for GNA-HHA (inclusive,Table 1).

Acknowledgements

This work comprised part of the studentship“Mannose-specific plant lectins from as diagnostics,vaccines and tools for the elucidation of bacterialand viral infection mechanisms”, a subsection of alarger project (Project Number FF821). The authors


acknowledge funding from the Scottish ExecutiveEnvironment and Rural Affairs Department.

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Novel mannose binding lectins from agricultural crops