the effect of peptide–pectin interactions on the gelation behaviour of a plant cell wall pectin

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Carbohydrate Research 335 (2001) 115–126

The effect of peptide–pectin interactions on the gelationbehaviour of a plant cell wall pectin

Alistair J. MacDougall,* Gary M. Brett, Victor J. Morris, Neil M. Rigby,Michael J. Ridout, Stephen G. Ring

Di�ision of Food Materials Science, Institute of Food Research, Norwich Research Park, Colney,Norwich NR4 7UA, UK

Received 30 November 2000; received in revised form 10 July 2001; accepted 30 July 2001

Abstract

The effect of basic peptides on the gelation of a pectin from the cell wall of tomato was examined through thedetermination of gel stiffness, and swelling behaviour of the gel in water. Poly-L-lysine, poly-L-arginine, and asynthetic peptide, designed to mimic a sequence of basic amino acids found in a plant cell wall extensin, act ascrosslinking agents. Circular dichroism studies on the interaction of synthetic extensin peptides with sodiumpolygalacturonate demonstrated that a conformational change was induced as a result of their complexation. Inaddition to their effect as crosslinking agents, the polycationic peptides reduced the swelling of the pectin networkin water. © 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Pectin; Extensin; Hydroxyproline-rich glycoprotein; Poly-L-lysine; Gelation

1. Introduction

The pectic polysaccharides are major com-ponents of the primary cell wall and middlelamella of dicotyledonous food plants.1 Struc-turally they consist of a backbone of (1�4)-�-D-galacturonosyl residues interrupted withtypically 10% substitution of (1�2)-�-L-rhamnopyranosyl residues. A portion of therhamnosyl residues are branch points for neu-tral sugar side-chains. Further structural di-versity is obtained from the methyl esterifi-cation of a portion of the galacturonosylresidues.1 They are understood to be predomi-nantly crosslinked in the cell wall throughionic interaction with calcium ions.2 Studies

on a pectin extracted from the cell wall mate-rial of unripe tomato, have shown that even atrelatively low concentrations (2–4% w/w) ofpolysaccharide, this ionic interaction wassufficiently strong for gelation to occur.3 Theextent to which these gels swelled in dilute saltsolution was governed by the balance betweengel stiffness and the affinity of the network forsolvent.4 The gels exhibited swelling behavioursimilar to that of other polyelectrolyte net-works, in which the affinity for solvent isprofoundly affected by the extent of dissocia-tion of the ionisable groups carried by thenetwork, and ionic strength. It was proposedthat although a major fraction of the unes-terified carboxyl groups in the pectin chainparticipated in ionic crosslinking, there was asufficient concentration of ‘free’ carboxylateto have a substantial effect on the observedswelling behaviour.

* Corresponding author: Fax: +44-1603-507723.E-mail address: [email protected] (A.J.

MacDougall).

0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 0 0 8 -6215 (01 )00221 -X

A.J. MacDougall et al. / Carbohydrate Research 335 (2001) 115–126116

Although a range of studies have examinedthe interaction of pectins with inorganiccations, relatively little work has been pub-lished on the possibility of structurally signifi-cant interactions occurring between pecticpolysaccharides and organic cationic com-pounds. The binding of polyamines to plantcell walls has been investigated,5 and it hasbeen suggested that these compounds mayhave a direct structural function in the cellwall.6 Dilute solution studies have also shownthat the interaction of basic peptides withpolygalacturonate can induce a conforma-tional change.7,8

Several different classes of structuralproteins have been identified in plant cellwalls, and prominent among these are thehydroxyproline-rich plant glycoproteins(HRGPs).9 HRGPs are basic proteins; theyare rich in hydroxyproline and serine, andare glycosylated mainly with arabinose andgalactose. In aqueous solution, HRGPs arereported to adopt a rod-like conformation,with a polyproline II helical structure pre-dominating.10,11 Early hypotheses concerningthe structural role of HRGPs concentratedon the possibility that covalent crosslinkingthrough tyrosine residues could lead to theformation of a protein network. There is evi-dence also for covalent linkage betweenHRGPs and pectin.12,13 At the same timethere has been consistent speculation thatHRGPs form ionic complexes withpectins.9,14,15

In this paper, we investigate the proposalthat ionic interactions between basic plantcell wall proteins and pectic polysaccharideshave the potential to contribute to the assem-bly of the cell wall network. The materialsselected for study were a HRGP from carrotroot (wound-induced extensin), and thechelator-extracted pectin from unripe tomatopericarp. The presence of clusters of basicamino acids in the amino acid sequence pre-dicted for carrot extensin16 appeared to fa-vour ionic interactions with pectin, andsynthetic peptides were used to examine theseinteractions more closely. Their behaviourwas compared to that of other basic peptides.The effect of these peptides on the crosslink-

ing and swelling behaviour of pectin gels wasexamined.

2. Experimental

Preparation of pectin.—Pectin was ex-tracted from purified tomato cell walls withtrans-1,2,diaminocyclohexane-N,N,N �,N �,-tet-raacetic acid (CDTA);3 concentrated pectinsolutions were prepared by rotary evapora-tion of stock solutions of the potassium salt,pH 6.5. The uronic acid and methyl estercontent of the pectic polysaccharide were de-termined as described previously.17

Preparation of extensin.—Extensin waspurified from carrot phloem parenchyma fol-lowing the procedure of Stafstrom and Stae-helin.18 Ion-exchange chromatography wascarried out on a 1.6×20 cm column of CMSephadex C50 (Pharmacia) equilibrated in 25mM Tris–HCl pH 8.0 at rt; the flow ratethrough the column was 30 mL/h. Carrotproteins were dissolved in 25 mL of bufferand were centrifuged for 15 min at 2500g toprecipitate insoluble material. After loadingthe protein solution, the column was washedwith buffer until the absorbance at 280 nmdropped to a constant value. The columnwas then eluted with a NaCl gradient from 0to 200 mM in 160 mL of buffer. Fractionsfrom the extensin-containing peak werepooled and subjected to diafiltration with 10vols of water, followed by freeze-drying. Thepurified protein was redissolved in distilledwater at a concentration of 20 mg/mL andstored at −20 °C. Analysis of the aminoacid content of the preparation was carriedout by the Protein and Nucleic Acid Chem-istry Facility, Cambridge University. Sensitiv-ity of the preparation to peroxidativecrosslinking was tested by exposure to a par-tially purified tomato extensin peroxidase.19

Approximately 60 �g of extensin protein wasincubated with extensin peroxidase in thepresence of 10 nmol of H2O2, in a total vol-ume of 100 �L of 0.1 M NaOAc buffer pH6.0. The elution profile of this mixture andthe separate proteins was determined by gel-filtration using a Pharmacia Superose-12column, eluted at 0.3 mL/min with 0.1 M

A.J. MacDougall et al. / Carbohydrate Research 335 (2001) 115–126 117

NaOAc, pH 5.0, 0.1 M NaCl, 0.02% sodiumazide, and UV detection at 280 nm.

Source and preparation of peptides.—Polyamino acids were obtained from Sigma.For poly-L-lysine the reported average-molec-ular weights (determined from viscosity) were1000, 2900, 3900 and 9800 Da, indicatingaverage dps of 6, 14, 19 and 47, respectively.Technical details provided by Sigma indicatedthat the dp of the 1000 Da material wasconfirmed by capillary electrophoresis; Mw/Mn

(SEC-LALLS) for the 9800 Da material was1.35. For poly-L-arginine the reported averageMw was 10,800 Da (dp 56) with Mw/Mn 1.1.Peptides were synthesised with a NovaSynCrystal (Calbiochem–Novabiochem, UK) au-tomatic peptide synthesiser using fluorenyl-methoxycarbonyl (Fmoc)–polyamide chem-istry, based on the method of Atherton andSheppard.20 Fmoc protected, activated aminoacids were purchased from Calbiochem–Nov-abiochem. After synthesis, peptides werecleaved from the polyamide resin support us-ing trifluoroacetic acid containing 5% (v/v)triisopropyl silane (Aldrich Chemical Co.) and5% (v/v) water as scavengers for the side-chainprotecting groups of the individual aminoacids. Scavengers were removed by Et2O ex-traction from aqueous solutions prior tofreeze-drying. Purity was assessed by C18 re-versed-phase HPLC, and fast-atom bombard-ment mass spectrometry (FAB-MS). Calciumcontamination was removed from synthesisedpeptides by gel filtration. A 1.6×98 cmcolumn of Biogel P2 ultrafine (Bio-Rad) wasequilibrated in glass distilled water and sam-ples, dissolved in 1 mL of water, were loadedon to the column under gravity. The columnwas eluted with distilled water at 15.5 mL/h atrt. Peptides were monitored by absorbance at280 nm and calcium by atomic absorbancespectrometry.

Formation of gels.—Gel formation was car-ried out at 4 °C. To form gels with basiccompounds, 350 mg of pectin solution(�1.5% w/w) were placed in a 2 mLstraight-sided (8 mm diameter) polythene mi-crocentrifuge tube, and 12 �L of 5.8% (w/v)HC1 was added to the surface. After 30 min,the basic compound was added in a totalvolume of 10–20 �L. After 24 h, 30 �L of 1 MNaOAc (pH 8) was added. Gels were removed

from the microcentrifuge tubes after a further24 h, and their physical properties were deter-mined after equilibration to rt. The final pHwas between 5.5 and 6.0.

Physical measurements of gels.—The stiff-ness of the gels was determined as the shearmodulus (G �) at 200 Hz calculated from themeasured velocity of a shear-wave passingthrough the gel using a Rank pulse shearome-ter.21 Gel swelling was measured either fromthe weight gain after 24 h, or from the changein linear dimensions of gel pieces of approxi-mately 1×1.5×4 mm. For the latter, gelswere formed in 40 mM CaCl2 and equilibratedwith 2 mL of 4 mM CaCl2 for 6 h (Stage 0).Gels were transferred to 1 mL of 4 mM CaCl2containing peptides and incubated for 16 h(Stage 1). The bathing solution was dilutedfourfold with distilled water (Stage 2) and thiswas repeated after two successive periods of24 h (Stages 3 and 4).

Circular dichroism.—Aqueous solutions ofsodium polygalacturonate (0.05% w/w) andpeptide (0.01% w/w) were prepared in waterpH 5.5–6.0. Spectra were recorded over therange 260–185 nm in a 0.2 cm cell using aJASCO 5-710 spectrophotometer, calibratedusing camphor sulphonic acid at 290 nm. Thescan speed was 100 nm/min and the reportedspectra are the mean of three scans. Dataanalysis was carried out using SELCONsoftware.

3. Results and discussion

Characterisation of synthesised peptides andextensin.—Two different peptides were syn-thesised from data for the genomic sequenceof carrot extensin.16 Peptide 1 (seven aminoacids) contained the sequence His–His–Tyr–Lys–Tyr–Lys. Peptide 2 (30 amino acids)contained this sequence and the additionalsequence His–His–Tyr–Lys–Tyr–Lys–Tyr–Lys (Fig. 1). Both peptides gave single peakson HPLC. With FAB-MS peptide 1 yieldedthe predicted molecular ion, [M+H]+, m/z=974.5. For peptide 2, mass determination wasby electrospray-ionisation mass spectrometry(ESI-MS). The major peak, which accountedfor over 70% of the material, was measured as3690.6�0.6 compared to a predicted molecu-


Fig. 1. Amino acid sequence for carrot extensin predicted by the genomic clone pDC5A1. The amino acid sequence given is fromthe data of Chen and Varner,16 with the putative signalling sequence underlined. The two peptides synthesised for use in this studyare given in bold type. Peptide 1 contains seven residues, and peptide 2 contains 30 residues.

lar weight of 3690. Two additional peaks,which accounted for the remainder of thematerial, were observed at 3619.6, and 3761.7,and represented the loss and gain of an ala-nine residue, respectively. This was consideredto be due to difficulties experienced in addingthe final residue (alanine), and not to be ofsignificance for the interpretation of the exper-imental data. Peptides were purified by gelfiltration to give a calcium content of �0.01�g calcium/mg solid.

Extensin was extracted from the cell wallsof aerated carrot root slices with calciumchloride.18 Extensin proteins were purified byion-exchange chromatography of thetrichloroacetic acid-soluble material in theseextracts (Fig. 2), with an overall yield of �10mg from 550 g of carrot phloem parenchyma.In carrots, two distinct forms of extensin (ex-tensin I and II) can be isolated.18 A restrictedset of fractions (6 mg) was therefore pooled(Fig. 2) for use in experiments. When chro-matographed by gel filtration (Fig. 3), thepurified protein eluted as a symmetrical majorpeak with a faster running minor peak, whichaccounted for 7% of the total peak area. Amarked decrease in elution time for the majorpeak was noted on pre-treatment with H2O2 inthe presence of a partially purified peroxidaseknown to specifically crosslink extensins (Fig.3).19 The molecular weight of the extensin, asdetermined by SELDI-MS (surface-enhancedlaser desorption ionization mass spectrome-try), was �71,000 g/mol. These data are con-sistent with the presence of a monomericextensin. The amino acid composition of thepurified extensin is given in Table 1, togetherwith the composition of extensin I and II, and

Fig. 2. Ion-exchange chromatogram of partially purified car-rot extensin. The dashed line indicates the salt gradient used,and the horizontal bar marks the fractions pooled for use inexperiments.

Fig. 3. Gel-filtration profile of carrot extensin, before andafter crosslinking with an extensin specific peroxidase. Beforecrosslinking, solid line; after crosslinking, dotted line; EP,peaks attributable to extensin peroxidase. The void volume ofthe column determined with Blue Dextran 2000 was 6 mL(retention time ca. 20 min).


Table 1Amino acid composition of carrot hydroxyproline-rich glycoproteins

Carrot HRGP (mol%)Amino acid Extensin 1 a (mol%) Extensin II a (mol%) Genomic clone pDC5A1 b (mol%)

46.5Hyp 27.550.0 –Asx 1.4 1.3 2.9 –

1.4 4.11.2 3.6Thr14.9 16.9Ser 10.911.9

2.6 6.00.2 2.6GlxPro 0.5 0.2 0.0 42.3

3.2 7.90.2 –Gly1.4 3.4Ala 1.40.40.0 0.0nd c –Cys5.5 3.7Val 4.06.00.2 0.0– 1.1Met0.5 1.0Ile –0.20.7 1.20.4 –Leu

4.5Tyr 10.5 3.9 12.00.3 0.0nd 0.7Phe

14.5His 5.8 10.5 9.57.3Lys 4.7 9.1 11.7

0.4 1.70.1 –ArgTrp – – – –

a Data from Ref. 31.b Data from Ref. 16.c Not detectable.

predicted data from the carrot genomic clonepDC5A1. The amino acid composition of thematerial is similar to that of extensin I, and topDC5A1 bearing in mind the post-transla-tional conversion of proline to hydroxypro-line. The low levels of threonine, glycine andalanine suggest that the level of extensin II inthe sample is low.

Effect of basic peptides and extensin onpectin gelation.—The pectic polysaccharideused in these studies was of the followingcomposition (mol%): GalA, 83; Gal, 12; Ara,3.2; Rha, 0.8; Glc, 0.6; Xyl, 0.1; Fuc, 0.1;Man, �0.1. The degree of methylesterifica-tion was 65%.3 A significant proportion of theunesterified galacturonic acid is presumed tobe distributed in a blockwise manner becauseof the ability of this pectin to form gels in thepresence of calcium ions. The pectin had ahigh intrinsic viscosity (810 mL/g) and gave abroad peak on size-exclusion chromatogra-phy.3 A wide range of different molecularlengths has been observed in a similarly ex-tracted tomato pectin which was examined byatomic-force microscopy.22 The basic peptidesand extensin preparation formed precipitateswhen mixed with concentrated pectin solu-

tions at neutral pH. This is consistent with thereported extent of interaction for mixtures ofpectin and gelatin23 or pectin and poly-L-lysine.7 Mixing of the basic peptides andproteins with concentrated solutions of pectinwas achieved by initial acidification of thepectin solution to a pH of �2 (50 mM HCl)in order to suppress the charge on the pectin.No measurable change in the level of methylesterification was observed at these pH valuesalthough the pectin did form a weak networkstructure. The basic components were thenallowed to perfuse into this network and thepH adjusted to 5.5–6.0.

Using this procedure it was possible to formelastic gels between pectin and a variety ofdifferent basic compounds, including thehighly basic peptides, poly-L-lysine (averagedp 6 or 19), poly-L-arginine (average dp 56).Fig. 4 shows the effect of the addition ofpoly-L-arginine (average dp 56) at 0.076, 0.14,0.28 and 0.43% (w/w) on the appearance of a1.3% (w/w) pectin gel. These additions corre-spond to charge ratios for peptide:pectin of0.22, 0.45, 0.90, and 1.37 respectively. All thegels are free standing. At initial levels of addi-tion, the resulting gel is essentially clear and


Fig. 4. Appearance of mixed pectin (1.3% w/w) poly-L-arginine gels (as a function of increasing peptide:pectin charge ratio (fromleft to right: 0.22, 0.45, 0.90, 1.37).

has the ability to recover from static deforma-tion. At higher levels a marked opacity, in-dicative of substantial polymer aggregation isapparent, coupled with a marked shrinkageand syneresis of the gel. Although peptide 1(dp 7) formed gels with pectin, peptide 2 (dp30) did not reliably form gels. In vivo carrotHRGP undergoes post-translational modifica-tion. None of the residues in peptide 1 areaffected in this way, but peptide 2 containsseveral proline residues which in the naturallyoccurring protein are converted to hydroxy-proline, with a portion of these being furthersubstituted with tetra-arabinoside side-chains.It is possible that the lack of these featureswill affect the interaction with pectin. In allcases, gelation could be reversed by leachingthe gels under the same acidic conditions usedto encourage mixing, demonstrating that theinteraction was non-covalent, and reversible,and that gel formation was not due to reduc-tive amination of the pectin.24 In addition, wefound that gels formed between peptide 1 andpectin were readily dispersed in 50 mMCDTA pH 6.5. This observation was of inter-est because CDTA is commonly used to ex-tract ionically bound pectin from plant cellwalls, and it demonstrates that CDTA candisrupt other ionic bonds crosslinking pectinsbesides those involving inorganic cations.

The shear modulus (G �) of the gels formedwith peptides or calcium ions was examined asa function of the molar concentration ofcrosslinking agent (Fig. 5). The most effectivecrosslinking agent for inducing gelation was

poly-L-arginine, followed by poly-L-lysine,peptide 1 and finally calcium ions. These datareflect the different affinities of the crosslink-ing agent for the pectin chain and suggest thatwhile a calcium crosslink is a cooperative as-sociation involving a number of calcium ions,a single multiply charged peptide can alsofunction as a crosslink. However the maxi-mum level of crosslinking in the peptide–pectin gels, as indicated by the shear modulus(�1100 N/m2, Fig. 5), is low compared to thelevel found in calcium–pectin gels preparedunder identical conditions at saturating levelsof calcium ions (2500 N/m2). This suggeststhat for the peptides there are a limited num-ber of potential crosslinking sites on the pectinbackbone. Peptide 1 induced a higher degreeof crosslinking than poly-L-lysine dp 6, despite

Fig. 5. Shear modulus as a function of the molar concentra-tion of added compound for gels formed with pectin (1.3%w/w) and different cations: �, poly-L-arginine dp 56; �,poly-L-lysine dp 6; �, peptide 1; �, calcium.


Fig. 6. Shear modulus of pectin gels (1.3% w/w) formed withmixtures of calcium and peptide 1 or peptide 2: �, no addedpeptide; �, plus peptide 1; �, plus peptide 2.

tion, the shear modulus of the gels was mea-sured (Fig. 6). These data show that in theabsence of added calcium ions, peptide 1formed gels, whereas peptide 2 did not, andthe lowest level at which calcium alone in-duced gel formation was 4 mM. At low levelsof added calcium, addition of either peptidewas more effective on a molar basis at increas-ing crosslinking than calcium alone. Thiseffect disappeared at higher levels of cross-linking. The increased level of crosslinking inthe mixtures could be either due to both thepeptide and the calcium crosslinking thepectin, or due to the peptide facilitating cal-cium mediated crosslinking. The absence ofany effect of added peptides on the ability of2 mM calcium to give increased crosslinking,suggests that crosslinking with both calciumand the peptide is occurring in the mixedsystems.

Although carrot extensin interacted at neu-tral pH (forming a precipitate) using themethod described above gel formation wasnot observed at levels of addition up to 10%(w/w) relative to pectin. We examined theeffect of pre-incubation of the pectin (1.3%w/w) with extensin on the properties of gelsformed after subsequent addition of 4 mMCaCl2 (giving a Ca:pectin charge ratio of 0.5).Under these conditions, maximum crosslink-ing occurs at 30 mM CaCl2 (a charge ratio of3.5). The results (Table 2) show that increas-ing the level of added extensin reduced theshear modulus of the mixed gels. This suggeststhat extensin is a poor crosslinking agent un-der these conditions and its interaction withthe pectin chain interferes with calciumcrosslinking.

Effect of basic peptides and extensin on theswelling beha�iour of pectin gels.—An addi-tional physical property of calcium pectin gelsis their tendency to swell when placed in dilutesalt solutions. In this respect they show be-haviour typical of polyelectrolyte networks,with swelling principally driven by an imbal-ance in the distribution of mobile counter ionsbetween the gel and the surrounding solution,which arises as the result of a Donnan effect.At equilibrium, the osmotic force drivingswelling is balanced by an elastic restoringforce resulting from the crosslinking of the

Table 2Stiffness and swelling of gels formed by addition of CaCl2 topectin pre-treated with carrot extensin

Weight of extensin Stiffness (G �) Volume change(N/m2)relative to pectin (%) on swelling (%)

0.2 +564853450.5 +59

1 365 +583502 +62

+653605+7428010

Extensin was mixed with a 1.3% w/w pectin solution byacidification as described in the Experimental Section. CaCl2was added after neutralisation to give a concentration of 4mM. Swelling was carried out by addition of 5 vols of water.Data for each treatment are the averages from the resultsfrom two gels.

having roughly half the charge (calculated onthe basis that at the pH of the gels histidineresidues do not contribute significantly to thecharge). The ability of peptide 1, to inducenetwork formation, despite its relatively low-charge density, suggests that non-ionic inter-actions can also contribute to the formation ofthe crosslink.

In order to further assess the interactionbetween peptide 1 or 2 and pectin in thepresence of calcium ions, mixtures containinga constant level of the peptide and increasingamounts of calcium were added to the acid-ified pectin solution (1.3% w/w). The chargeratio of peptide:pectin in these experimentswas 0.14 for peptide 1 and 0.11 for peptide 2.After incubation and subsequent neutralisa-


polymer network. The swelling pressure canbe estimated from the relationship,25

�=RT(ic2)2/2w�c s*

where R is the gas constant, T is the tempera-ture in Kelvin, i is the degree of ionisationmultiplied by the valency of the charge on thepolymer, c2 is the concentration of polymercharge expressed as moles of repeating unit, wis the valency factor of the electrolyte, � is thenumber of cations and anions into which theelectrolyte dissociates, and c s* is the concentra-tion of strong electrolyte external to the gel.Swelling is influenced by the extent of ionisa-tion of the polymer and the valency of thecounterion. Counterions of higher valency re-duce the swelling pressure.

The effect of addition of peptides on theswelling of mixed pectin gels prepared withpoly-L-lysine dp 6, or poly-L-arginine dp 56and pectin is shown in Table 3. Although theshear modulus of the gels formed with differ-ent amounts of each compound was similar,indicating that there is a similar level ofcrosslinking of the pectin matrix, the subse-quent swelling behaviour showed a markeddependence on the amount of added peptide.At higher levels of added peptide reducedswelling was observed and ultimately, forpoly-L-arginine, shrinkage—which is a featurenot found in pectin gels formed with equiva-lent levels of calcium. At swelling equilibrium

the osmotic pressure driving swelling and themodulus are equivalent.26 Variation in theshear modulus at swelling equilibrium (Table3) therefore reflects the effect of complex for-mation on the extent of ionisation of thepectin network, and the effect of peptide mul-tivalency on the Donnan equilibrium. Esti-mates of the extent of ionisation of the pectinnetwork for gels formed with poly-L-arginineat a charge ratio of 0.22 were made, assumingthe added peptide is fully complexed with thepectin, but not allowing for loss of pectinfrom the gel during swelling. Dissociation ofless than 50% of the remaining ionisablegroups is required to explain the observedswelling modulus. This in turn would be con-sistent with a blockwise distribution of thecharge on the pectin leading to reduced ionisa-tion through counter-ion condensation.27

In a related experiment, the effect of pep-tides on the swelling of calcium–pectin gelnetworks was observed. Gels were formed in40 mM CaC12 and were allowed to reachswelling equilibrium in 4 mM CaC12; pectinwhich did not participate in network forma-tion was removed at this stage.3 The gels werethen placed in 4 mM CaCl2 in the presence ofdifferent amounts of peptide, and were sub-jected to a progressively increased swellingpressure by successive dilution of the bathingsolution (Fig. 7). The effectiveness of addedpeptides in reducing swelling broadly reflects

Table 3Stiffness and swelling of peptide–pectin gels

Weight relative toPeptide Charge ratio Stiffness after swelling (G �)Stiffness (G �) Volume change on(N/m2)pectin (%) swelling (%)peptide:pectin (N/m2)

6Poly-L-arginine 18250.22 +28102011(average dp 56) +2410800.45 232522 0.90 1190 −19 164034 1.37 1082 −14 17356 0.31Poly-L-lysine 230 +49 265

213 +41(average dp 6) 42511 0.631.25 264 +24 350221.9034 242 +1 355

Gels were formed as described in the Experimental Section using a pectin solution of 1.3% (w/w). The amount of peptide addedis presented both as the weight relative to the pectin and the charge relative to pectin assuming full ionisation of both compounds.Swelling was carried out by placing the gels (approximately 250 mg) in 4 mL of distilled water. Data for each treatment are theaverages from two gels.


Fig. 7. Swelling of calcium pectin gels on successive dilutionafter treatment with basic peptides. Stage 0, no addition;Stage 1, addition of peptide; Stages 2, 3 and 4, successivefourfold dilutions of the bathing solution with distilled water.The amounts of peptide added as a proportion of the totalpectin present were: �, no peptide added (control); �, 2; �,5; �, 10; �, 20; , 40% (w/w). The peptide:pectin chargeratio for each treatment is given on the graph.

terified residues, which account for 35% of thetotal galacturonic acid, are believed to bemainly distributed in blocks.3 The interactionof basic peptides with sequences of unes-terified uronic acid residues was probedthrough examination of the circular dichroism(CD) of dilute aqueous solutions of polygalac-turonic acid–peptide mixtures. The change inCD of polygalacturonic acid (0.05% w/w) onaddition of calcium ions (5 mM) is shown inFig. 8(a). The observed change is consistentwith previous observations29 and has been in-terpreted as a perturbation in the carboxylateabsorbance as a result of ion binding. Formixtures of peptides (0.01% w/w) and poly-galacturonic acid (0.05% w/w), the observedbehaviour is compared with that predictedfrom summation of the spectra of the separatecomponents. The poly-L-lysine–polygalactur-onic acid interaction was examined as a func-tion of dp of the poly-L-lysine. For dps 6, 14and 19 there was no marked difference inpredicted and observed behaviour (Fig. 8(b–d)), indicating that the poly-L-lysine crosslink-ing of the pectic polysaccharide network doesnot necessarily induce a conformationalchange. At the higher dp of 47 (Fig. 8(e)),there was a difference in predicted and ob-served behaviour indicative of conformationalchange and consistent with published observa-tion.7,8 The CD spectrum for peptides 1 and 2(Fig. 9(a)) were analysed by SELCON curvefitting. The analysis of peptide 1 did not pro-duce reliable results but peptide 2 was fitted at(P�0.01) for a composition of 4% � helix,46% � sheet, 29% � turn, with 21% unas-signed. Fitting for the polyproline II helix,which is likely to be the conformation of theadjacent proline residues, was not included inthe analysis and may account for part of theunassigned data.30 On addition of polygalac-turonic acid (0.05% w/w) to peptides 1 and 2(0.01% w/w) a spectral change is observed(Fig. 9(b,c)). This indicates that complexationof these peptides with polygalacturonic acidinduces a conformational change. The evi-dence for conformational change on mixingpeptide 1 and polygalacturonic acid stands incontrast to the data for poly-L-lysineoligomers of similar dp where no spectralchange is observed. A specific interaction be-

the peptide:pectin charge ratio. These datapartly reflect the ability of the peptides tocrosslink the pectin network, but may alsoresult from suppression of polymer ionisationand the valency of the peptide as describedabove. These effects of the peptides are ofinterest because of the potential physiologicalsignificance of pectin hydration for plant cellwall behaviour in vivo.28

Effect of basic peptides and extensin on thesolution conformation of pectin.—In thetomato pectin used in this study, the unes-


Fig. 8.

tween peptide 1 and 2 and polygalacturonicacid is indicated. It is not possible to tell fromthese data alone, however, whether the con-formational change is in the polygalacturonicacid, the peptide or both.

The conclusion that there is a specific inter-action between peptide 1 and polygalacturonicacid is of interest in understanding the role ofthe peptide motif Tyr–Lys–Tyr–Lys. Thishas been identified as a potential site for cova-lent inter- and intramolecular crosslinkingthrough the formation of isodityrosine.15 Thedata we have obtained can be interpreted asproviding an alternative function for thissequence, or as indicating that isodityrosineformation is likely to be affected bycomplexation of extensins with pecticpolysaccharides.

4. Conclusions

Oligomeric basic peptides have the potentialto act as non-covalent crosslinking agents inpectin networks. A basic, oligomeric, frag-ment of a cell wall extensin can also functionin this way. A synergy between calcium-medi-ated crosslinking and peptide crosslinking wasobserved under some conditions. In additionto their effect on crosslinking, basic peptideswithin the pectin network can have a markedeffect on the swelling behaviour of the gel.This is partly due to the inclusion of a multi-valent counterion, and is also partly a result ofspecific interactions of the peptide with thepectin chain reducing its effective charge. Aplant cell wall carrot extensin does interactnon-covalently with pectin, and this interac-tion can be explained as the outcome of spe-cific binding to the pectin of particular basicamino acid sequences present in the protein.The data support the contention that non-co-valent interactions anchor the extensin to the

Fig. 8. CD spectra for 0.05% (w/w) polygalacturonic acidcomplexed with calcium (a), or 0.01% (w/w) poly-L-lysine, dp6 (b), dp 14 (c), dp 19 (d), dp 47 (e). For mixtures ofpolygalacturonic acid and poly-L-lysine the observed spec-trum (solid line) is compared with a predicted spectrumobtained from the sum of the two components analysedseparately (short dash). For dp 6 (b) the separate spectra forpolygalacturonic acid (long dash), and poly-L-lysine (shortand long dash) are included.


Fig. 9. CD spectra for; (a) peptide 1 and 2; (b) peptide 1mixed with polygalacturonic acid; (c) peptide 2 mixed withpolygalacturonic acid. Peptides were at 0.01% (w/w) andpolygalacturonic acid at 0.05% (w/w). For mixtures (b,c) theobserved spectrum (solid line) is compared with a predictedspectrum obtained from the sum of the two componentsanalysed separately (short dash).

for carrying out the SELDI-MS. The authorsthank the BBSRC core strategic grant forfinancial support.

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the effect of peptide–pectin interactions on the gelation behaviour of a plant cell wall pectin

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