Carbohydrate Research 345 (2010) 2648–2656

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Carbohydrate Research

journal homepage: www.elsevier .com/locate /carres

Carbohydrate structural analysis of wheat flour arabinogalactan protein

Theodora Tryfona a,b, Hui-Chung Liang a, Toshihisa Kotake c, Satoshi Kaneko d, Justin Marsh e,Hitomi Ichinose d, Alison Lovegrove e, Yoichi Tsumuraya c, Peter R. Shewry e, Elaine Stephens b,�,Paul Dupree a,⇑a School of Biological Sciences, Department of Biochemistry, Hopkins Building, The Downing site, Tennis Court Road, Cambridge University, CB2 1QW Cambridge, UKb School of Physical Sciences, Department of Chemistry, University Chemical Laboratory, Lensfield Road, Cambridge University, CB2 1EW Cambridge, UKc Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japand National Food Research Institute National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japane Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK

a r t i c l e i n f o

Article history:Received 28 May 2010Received in revised form 14 September2010Accepted 16 September 2010Available online 21 September 2010

Keywords:Arabinogalactan protein (AGP)ArabinopyranoseWheat flourMALDI-CIDAG hydrolytic enzymes

0008-6215/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.carres.2010.09.018

⇑ Corresponding author. Tel: +44 1223 333340; faxE-mail address: p.dupree@bioc.cam.ac.uk (P. Dupr

� Present address: Laboratory of Molecular Biology0QH, UK.

a b s t r a c t

The water-extractable arabinogalactan protein (AGP) was isolated from bread wheat flour (Triticum aes-tivum L. variety Cadenza) and the structure of the arabinogalactan (AG) carbohydrate component wasstudied. Oligosaccharides, released by hydrolysis of the AG with a range of AGP-specific enzymes, werecharacterised by Matrix Assisted Laser Desorption Ionisation (MALDI)-Time of Flight (ToF)-Mass Spec-trometry (MS), MALDI-ToF/ToF high energy collision induced dissociation (CID) and Polysaccharide Anal-ysis by Carbohydrate gel Electrophoresis (PACE). The AG is composed of a b-(1?3)-D-galactan backbonewith b-(1?6)-D-galactan side chains. These side chains are highly variable in length, from one to at least20 Gal residues and are highly substituted with a-L-Araf. Single GlcA residues are also present at the non-reducing termini of some short b-(1?6)-galactan side chains. In addition, the b-(1?6)-galactan sidechains are also substituted with b-L-Arap. We propose a polysaccharide structure of the wheat flourAGP that is substantially revised from earlier models.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Arabinogalactans (AGs) are structurally complex large branchedpolysaccharides, attached to hydroxyproline (Hyp) residues ofmany plant cell wall polypeptides. Proteins glycosylated with AG(AGPs) have been attributed a range of functions such as develop-mental regulation, adhesion, wound healing and salt or droughtresistance.1 The AG may confer properties on the glycosylated pro-tein such as enhanced secretion efficiency or stability in the cellwall.2 Although most AGPs have both AG glycosylated domains(glycomodules) and structural or enzymatic domains, many consistof over 90% polysaccharide and have a small peptide core, suggest-ing that the AG is the functional part of the molecule.1–5 The gly-cosylated domain of the AGP is typically rich in Hyp, serine (Ser),alanine (Ala) and threonine (Thr) and is resistant to proteolysispresumably due to its substitution with bulky carbohydrategroups.6 The carbohydrate moieties in many AGPs, known as typeII AG, have been shown to consist of b-(1?3)-, b-(1?6)- andb-(1?3)-b-(1?6)-linked D-galactopyranosyl (D-Galp) residues.7

ll rights reserved.

: +44 1223 333345.ee)., Hills Road, Cambridge CB2

The Gal can be further substituted with L-arabinofuranosyl (L-Araf)and occasionally also L-rhamnosyl (L-Rha), L-fucosyl (L-Fuc) andD-glucuronosyl (D-GlcA) (with or without 4-O-methylation) resi-dues. The AG may consist of as many as 100–120 sugar units.8

AG shows enormous heterogeneity in size and composition, mak-ing structural determination difficult.9 Thus, AGPs may be the moststructurally complex molecules in nature and so far no completestructure of a native AGP has been elucidated.

The non-starch polysaccharides of wheat flour consist mainly ofarabinoxylan (AX), b-(1?3) (1?4)-D-glucan (b-glucan) and a sin-gle species of water-extractable AGP7,10 which can be isolated byan ethanol precipitation method described by Loosveld et al.11,12

and Fincher et al.7 This wheat endosperm AGP has been estimatedto be present at a level of 0.27–0.38% of the flour dry weight andshown to influence the bread making properties.13 The reportedaverage mass ranges from 22 kDa7,14,15 to 70 kDa12 depending onthe measurement techniques used.16 The AGP has a peptide back-bone considerably smaller than most AGPs,15 typically composedof 15 amino acids with Hyp, Ala, Ser and Thr being the most abun-dant. The AGP has been isolated from many different wheat varie-ties and there is very little sequence variation in the core peptide.12

The sequence of the peptide is identical to that of the grain softnessprotein.16 The polysaccharide portion of the wheat AGP constitutesover 90% of the molecular mass.

T. Tryfona et al. / Carbohydrate Research 345 (2010) 2648–2656 2649

Early reports of the structure of the wheat flour AG date back to1974.7 NMR spectroscopy and methylation analyses suggested theAG consists of a b-(1?6)-Galp backbone, highly substituted withsingle a-L-Araf residues at C-3 of the Gal, with no terminal Gal res-idues.12,17 Further NMR and methylation studies elaborated thismodel by suggesting that the b-(1?6)-Galp backbone can also besubstituted with a single b-Gal at C-3, which may in turn be substi-tuted at C-3 with a single a-L-Araf residue.16

The proposed carbohydrate structure of the wheat flour AGP isdifferent from the most frequently reported structure for AG,which is a b-(1?3)-Galp backbone with short b-(1?6)-Galp sidechains.1 These side chains can carry single a-(1?3)-L-Araf substi-tutions linked at C-3 and GlcA or 4-O-methyl-GlcA (4-Me-GlcA)at the non-reducing termini linked at C-6. The b-(1?3)-Gal back-bone may be interspersed with occasional periodate oxidation sen-sitive ‘kinks’ of unknown nature.18,19 Consistent with this model,Tan et al. have reported a structure of a small AG from a syntheticAGP expressed in tobacco, which consisted of a Hyp-linked b-(1?3)-Gal pentasaccharide backbone with a single b-(1?6)-Gal‘kink’.20 This backbone had single b-(1?6)-Gal side chains substi-tuted at C-3 with single a-L-Araf or short a-L-Araf oligosaccharidechains and at C-6 with GlcA or L-Rha-GlcA.20

Even though the structures of some AGs have been determinedby NMR and linkage analyses, the complete structural elucidationof a native AG still remains a formidable task, not only becauseNMR requires purified samples in high quantities but also becauseAG molecules are notorious for their structural complexity andheterogeneity. NMR spectroscopy and methylation analysis havebeen largely used to provide information regarding the amountand type of linkages between adjacent glycosyl residues, and het-erogeneity can confound attempts to build complete structuralmodels. Recently, enzymes able to release oligosaccharides specif-ically from AG have been described.21–24 These enzymes allow newapproaches to AG structural determination to be attempted.21 Highenergy Matrix Assisted Laser Desorption Ionisation (MALDI)-Collision Induced Dissociation (CID) mass spectrometry can pro-vide extensive structural information on oligosaccharides and hasbeen used to determine details of the sequence, branching andlinkage of AX isomers25 and to characterise N-glycan structures re-leased from honey bee phospholipase A2 and Arabidopsis thalianaglycoproteins.26 Polysaccharide Analysis by Carbohydrate gel Elec-trophoresis (PACE) has also been used extensively for characterisa-tion of plant polysaccharide structures and its abundance.27 In thisstudy the carbohydrate component of the wheat flour AGP isolatedfrom water extracts of wheat endosperm with ethanol precipita-tion as previously described7,11,12,16 was studied. AG-specificenzyme digestion products were analysed by PACE and MS,allowing a revised partial structure to be proposed.

2. Results and discussion

2.1. Wheat flour AGP is sensitive to digestion by AG specificenzymes

We first investigated whether the AG of wheat flour AGP pos-sesses any b-(1?3)-galactan susceptible to exo-b-(1?3)-galactanase hydrolysis. Monosaccharide analysis by High pH AnionExchange Chromatography (HPAEC) with Pulsed AmperometricDetection (PAD) of crude Cadenza wheat flour AGP confirmed asimple L-Ara and Gal composition with a molar ratio of 0.5 whichis consistent with previous reports (0.63–0.72) (data notshown).12,15 A small amount of GlcA was also detected, and thisfinding is discussed later (Section 2.2). The wheat AGP was incu-bated with exo-b-(1?3)-galactanase, and the resulting oligosac-charides were analysed by PACE. Oligosaccharides of different

degrees of polymerisation (DP), linkage or composition areresolved by the gel electrophoresis, and band intensity is propor-tional to the quantity of oligosaccharide. Since exo-b-(1?3)-galactanase specifically cleaves terminal b-(1?3)-galactosidic link-ages and can bypass branching points, it can liberate any b-(1?6)-linked galactosyl side chains in the AG as oligomers.22 From Figure1a (lane 2) it is clear that the wheat flour AGP is susceptible toexo-b-(1?3)-galactanase, indicating the presence of a b-(1?3)-galactan. The released sugars included Gal and diverse oligosaccha-rides including products co-migrating with b-(1?6)-galactobiose.In order to evaluate the effect of a-L-Araf substitution on theexo-b-(1?3)-galactanase digestion, the AGP was incubated witha-L-arabinofuranosidase, and then digested with the exo-b-(1?3)-galactanase. The released oligosaccharide fragments were analysedby PACE (Fig. 1a, lane 3). Many of the oligosaccharides migratingslower than b-(1?6)-Gal3 were missing from the a-L-arabinofura-nosidase plus exo-b-(1?3)-galactanase treatment, and a widerange of new and diverse oligosaccharide fragments appeared,resulting in a shift of the banding pattern. This suggests that manyof the products of the exo-b-(1?3)-galactanase are substitutedwith a-L-Araf residues. A clear ladder of oligosaccharides withDP extending from 1 to over 20 was visible. The intensity andnumber of bands were increased after a-L-arabinofuranosidasetreatment, indicating that a higher quantity of oligosaccharideswas released. The results demonstrate that pre-treatment witha-L-arabinofuranosidase enhances the exo-b-(1?3)-galactanasedigestion. A Yariv gel diffusion assay showed that the weak Yarivreactivity of the wheat flour AGP is lost after treatment with AGP-hydrolysing enzymes, confirming that the bulk of the AGP wassensitive to enzymatic digestion (Fig. 2).

To determine the mass of the released oligosaccharides, thesame enzyme digested samples were per-methylated and analysedby MALDI-ToF-MS (Fig. 1b). The oligosaccharides released by exo-b-(1?3)-galactanase consist predominantly of hexosyl and pento-syl residues. Based on the compositional data of the AGP these areD-Gal and L-Ara. Moreover, the a-L-arabinofuranosidase removed alarge proportion of the pentosyl residues, leaving a putative galac-tan series of DP 2 to DP 9 (Fig. 1c). In addition, we were able to de-tect ions with m/z values that correspond to short oligosaccharidescarrying (Me)-hexuronosyl residues (m/z 695.4 and 855.6).

To determine the structure of the exo-b-(1?3)-galactanase re-leased oligosaccharides, we carried out MALDI-CID of a Hex3Pent2

(m/z 1001.8) oligosaccharide (Fig. 3a). The series of 1,5X and Y ionsshow the distribution of Gal residues, whilst the non-reducing end0,4A3 ion confirms the sequence and also indicates the presence of(1?6) glycosidic linkages between two of the Gal residues. G3

‘elimination ions’ provide information about the L-Ara residues,indicating that they are likely to be present in their furanose form,whilst another important ‘elimination ion’ (D) shows the presenceof an L-Araf at the C-3 of Gal residues. This oligosaccharide wastherefore identified as a-L-Araf-(1?3)-b-Galp-(1?6)-[a-L-Araf-(1?3)]-b-Galp-(1?6)-Galp. This structure is consistent with itbeing a b-(1?6)-galactan side chain released from a b-(1?3)-galactan backbone by the exo-b-(1?3)-galactanase.6,28 Similarly,an oligosaccharide product of the a-L-arabinofuranosidase andexo-b-(1?3)-galactanase sequential digestion (m/z 1089.7 massof Hex5), was analysed by MALDI-CID (Fig. 3b). The presence of acomplete series of 1,5X ions differing by m/z 204 (a hexose residue)in the MALDI-CID spectrum suggested that the chain is linear, pos-sibly with (1?6)-links. The presence of the 0,4A and 3,5A ions,which are unique to the non-reducing end, confirm this latterassumption. The oligosaccharide was identified as b-(1?6)-galactopentaose. Taken together, the PACE and MS characterisationof the enzyme products suggests that the wheat AG contains longb-(1?6)-galactan side chains on a b-(1?3)-galactan backbone.

464 812 1160 1508 1856Mass (m/z)

0102030405060708090

100

% I

nten

sity

477.

3

681.

569

5.4

841.

6

885.

6

1045

.710

89.7

1249

.812

93.9

1453

.914

09.9

1614

.016

58.0

1818

.1

1702

.1

1570

.0

1906

.2

855.

6

464 812 1160 1508 18560

102030405060708090

100

% I

nten

sity

477.

4

637.

5

1001

.8

841.

7

1366

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1162

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1730

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1570

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2094

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Hex3Pent

Hex3Pent

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3

Hex4Pent

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1730

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2094

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1934

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4

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4

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5

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xeH

3Pen

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1249

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1453

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Hex6

Hex8 Hex

9

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5tneP2

Hex6Pent

Hex7Pent

xeH

6Pen

t 2

xeH

7tneP2

1 2 3(a)

(c)

(b)

Ara

Gal

(Gal)3

(Gal)2

(Gal)4

Hex

714

98.0

Mass (m/z)

Figure 1. Oligosaccharides released by AG specific enzymes analysed by (a) PACE. 1, L-Ara and b-(1?6)-galactooligosaccharide standards. AGP digested with: 2, exo-b-(1?3)-galactanase; 3, a-L-arabinofuranosidase followed by exo-b-(1?3)-galactanase; (b) and (c) MALDI-ToF-MS spectra of per-methylated oligosaccharides released by exo-b-(1?3)-galactanase or a-L-arabinofuranosidase followed by exo-b-(1?3)-galactanase, respectively. Peaks marked with a (*) were selected for MALDI-CID structural analysis.The insets show a magnified region of the spectrum.

Figure 2. Yariv gel-diffusion assay of AGPs. A, buffer; B, gum Arabic (positivecontrol); C, oat spelt xylan (negative control); D, crude wheat flour AGP; E, wheatflour AGP hydrolysed with a-L-arabinofuranosidase (x2 digestion), exo-b-(1?3)-galactanase, b-glucuronidase, endo-b-(1?6)-galactanase, b-L-arabinopyranosidase,a-L-arabinofuranosidase and endo-b-(1?6)-galactanase; F, buffer.

2650 T. Tryfona et al. / Carbohydrate Research 345 (2010) 2648–2656

2.2. Wheat flour AG carries glucuronosyl substitutions

The MALDI-ToF-MS spectra of a-L-arabinofuranosidase and exo-b-(1?3)-galactanase combined digestion of wheat AGP indicatedthe presence of previously unreported short AG side chain frag-ments carrying hexuronosyl substitutions (m/z 695.4 and 855.6;Fig. 1c). As discussed above, monosaccharide analysis of acidic sug-ars had also revealed that wheat AGP carried GlcA. In order to ver-ify the presence of GlcA on the exo-b-(1?3)-galactanase releasedoligosaccharide fragments, the samples were subjected to incuba-tion with an AG-specific b-glucuronidase,29 followed by PACE andMALDI-ToF-MS analysis. PACE showed an alteration on thefingerprint after b-glucuronidase digestion, indicating some oligo-saccharides were susceptible to b-glucuronidase (Fig. 4a). TheMALDI-ToF-MS spectra also indicated the absence, after b-glucu-ronidase hydrolysis, of m/z 695.4 and 855.6 ions correspondingto Gal2(Me)GlcA and Gal2Ara(Me)GlcA, respectively (Fig. 4b). Fur-ther structural characterisation by high energy MALDI-CID of theion at m/z 695.4 showed that GlcA is linked to the C(O)6 positionof the short b-(1?6)-galactan side chains (Fig. 4c). Since the oligo-saccharides are released by the exo-b-(1?3)-galactanase, thereducing end must be Gal, and we therefore conclude that the GlcA

150.0 331.6 513.2 694.8 876.4Mass (m/z)

827.

285

5.3

925.

2623.

365

1.3

461.

3

811.

2

489.

3

287.

3

779.

3

591.

3

939.

3

419.

3

1001

.4

371.

3

449.

3

227.

2

259.

3

735.

3

561.

4

401.

3

O

O

O

O

O

O

O

O

O

D1

Y1

1,5X1

E2 B

2

C2

W2 0,4A

3

3,5A3

H2

D2

Y2a

1,5X2α

E3

G2α

W3

Y3

Y2β

1,5X3

1,5X2β

E4

G4

G2α

1,5X3

1,5X2β

150.0 350.2 550.4 750.6 950.8Mass (m/z)

463.

4

1089

.4

491.

3

695.

366

7.4

899.

3

709.

4

871.

3

839.

3

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533.

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635.

4

415.

3

913.

3

287.

3

605.

4

809.

3

259.

3

329.

3

211.

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O

O

O

O

O

E1

Y1

1,5X1

3,5A2

H2

E2

Y2

1,5X2

0,4A33,5A

3H

3

E3

D3

Y3

1,5X3 0,4A

4

3,5A4 H

4

D4

Y4

1,5X4

0,4A5

E1

E2

E3

1,5X1

1,5X2

1,5X3

1,5X4

3,5A2

3,5A3

3,5A4

0,4A4

0,4A3

D3

D4

Y1

Y3

Y2

Y4

H4

H3

H2

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[M + Na]+

[M + Na]+

E4

E3

E2

G4

G3β

Y2β

1,5X1

1,5X2α

D1

D2

B2

W3

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W2

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3

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OCH3

OCH3

OCH3

OCH3

H3CO

H3CO

H3CO

H3CO

H3CO

CH2

H3COH

2C

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CH2

CH2OCH

3

OCH3

OCH3

OCH3

OCH3

OCH3

H3CO

H3CO

H3CO

H3CO

H3CO

H3CO

H3CO

H3CO

H3CO

H3CO

CH2

CH2

CH2

Figure 3. MALDI-CID of (a) a Hex3Pent2 oligosaccharide released by exo-b-(1?3) galactanase and (b) a Hex5 oligosaccharide released by combined a-L-arabinofuranosidaseand exo-b-(1?3)-galactanase hydrolysis. Glycosidic and cross-ring fragments are identified according to Domon and Costello nomenclature.46

T. Tryfona et al. / Carbohydrate Research 345 (2010) 2648–2656 2651

is present on their non-reducing ends. The oligosaccharide wasthus identified as b-GlcA-(1?6)-b-Galp-(1?6)-b-Galp.

Both GlcA20,30,31 and 4-Me-GlcA8,21,32 have been reported inAGPs but MALDI-ToF-MS after per-methylation of oligosaccharidesdoes not distinguish these two residues. We therefore performedperdeutero-methylation, a classical approach to distinguish be-tween methylated and non-methylated forms of sugars. MALDI-ToF-MS analysis of perdeutero-methylated oligosaccharides ofAGP hydrolysed by a-L-arabinofuranosidase and exo-b-(1?3)-galactanase, is shown in Figure 4d. Signals corresponding to

Gal2GlcA and Gal2AraGlcA (m/z 728.5 and 894.6, respectively) weredetected whilst m/z 725 and 891 signals corresponding toGal2MeGlcA and Gal2AraMeGlcA were absent from the spectra.Thus, wheat flour AGP contains short oligosaccharides terminatingin GlcA, and no 4-Me-GlcA residues were detected.

2.3. Wheat flour AGP carries arabinopyranosyl residues

Endo-b-(1?6)-galactanase hydrolyses b-(1?6)-galacto oligo-mers with DP 3 or higher, producing Gal and galactobiose

650 700 750 800 850 900Mass (m/z)

0

10

20

30

40

50

60

70

80

90

100

% I

nten

sity

681.

5

697.

5

885.

6

841.

6

857.

6

Gal3

[M + Na]+

Gal3

[M + K]+

Gal3Ara

[M + Na]+

[M + K]+

Gal3Ara [M + Na]+

Gal4

(b)

150 267 384 501 618Mass (m/z)

463.

2

287.

2

519.

2

343.

2

605.

2

491.

2

401.

2

315.

2

445.

2

211.

2

415.

3

385.

3

431.

2

259.

2

591.

2

241.

2

273.

2

477.

2

549.

2

695.

2O

O

O

O

O

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H3CO

H3CO

H3CO

H3CO

CH2

CH2

COOCH3

G1 Z

1

D1

Y1C

1

1,5X1

0,4A2

0,2X1

3,5A2

G2

H2

1,5A2D

2Z

2

Y2

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0,2X2

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3

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3

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C1

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0,2X2 1,5X

1

(c)

(a)1 2

Ara

Gal

(Gal)3

(Gal)2

(Gal)4

*

700 742 784 826 868 910Mass (m/z)

0

10

20

30

40

50

60

70

80

90

100

% I

nten

sity

714.

5

728.

5

880.

6

894.

6

Gal3

Gal2GlcA

Gal3Ara

Gal2AraGlcA

(d)

OCH3

OCH3OCH

3

OCH3

H3CO

H3CO

Figure 4. b-Glucuronidase-sensitive oligosaccharides (a) revealed by PACE are shown by (*). AGP digested with: 1, a-L-arabinofuranosidase followed by exo-b-(1?3)-galactanase (as shown in Fig. 3); 2, a-L-arabinofuranosidase, exo-b-(1?3)-galactanase and b-glucuronidase. (b) MALDI-ToF-MS spectrum of per-methylated oligosaccharidesreleased by a-L-arabinofuranosidase, exo-b-(1?3)-galactanase and b-glucuronidase digestion. (c) MALDI-CID of the Gal2GlcA oligosaccharide released by sequential digestionof AGP with a-L-arabinofuranosidase and exo-b-(1?3)-galactanase. (d) MALDI-ToF-MS spectrum of perdeutero-methylated oligosaccharides released by sequential digestionwith a-L-arabinofuranosidase and exo-b-(1?3)-galactanase.

2652 T. Tryfona et al. / Carbohydrate Research 345 (2010) 2648–2656

fragments, but it is intolerant of substitution with a-L-Araf resi-dues.33 We showed above that the oligosaccharides released bythe exo-b-(1?3)-galactanase hydrolysis of wheat AGP are substi-tuted b-(1?6)-galactans. These side chains should therefore be

susceptible to endo-b-(1?6)-galactanase digestion, and afterexhaustive treatment with a-L-arabinofuranosidase, b-glucuroni-dase and endo-b-(1?6)-galactanase should be hydrolysed torelease Gal and b-(1?6)-galactobiose fragments. However, any

T. Tryfona et al. / Carbohydrate Research 345 (2010) 2648–2656 2653

additional types of substitution on the side chains may also pre-vent complete hydrolysis by the enzyme. Hydrolysis of thestripped side chains with endo-b-(1?6)-galactanase indeed re-leased Gal and b-(1?6)-galactobiose (Fig. 5a, lane 2). However, aladder of large additional oligosaccharide fragments remained.MALDI-ToF-MS analysis revealed that galactooligosaccharides car-rying between one and three L-Ara were still present in the sample(Fig. 5b). In order to investigate the linkage of these L-Ara residuesto the galactan chain, the Gal4Ara oligosaccharide with the stron-gest signal on the MS spectrum (m/z 1045.3) was selected for MAL-DI-CID structural analysis (Fig. 5c). This revealed that the L-Ara was(1?3)-linked to the third Gal from the reducing end of the oligosac-

459.0 841.80

10

20

30

40

50

60

70

80

90

100

% I

nten

sity

477.

2

1045

.3

841.

2

637.

2

885.

2

681.

2

Gal2

Gal2Ara

Gal3

Gal3Ara

Gal4

Gal4A*

G

150.0 340.8 531.6

491.

346

3.3

287.

225

9.2

431.

3

329.

3

519.

3

211.

2

401.

3

227.

2

O

O

O

O O

O

E1 D

1

Y1

1,5X1

0,4A2

3,5A2

1,5X2Y

2

H2

D2 0,2X

2 E

(a)

Ara

Gal

(Gal)3

(Gal)2

(Gal)4

G4

Y3α

H4

H

0,4A2

1,5X3α

3,5A2 Y

3βH3β

0,2X3,5A

1,5X3β

Araf

Arap

OCH3

OCH3

OCH3

OCH3

OCH3

H3CO

H3CO

H3OC

H3CO

CH2OCH

3

CH2

OCH3

H3COH

2C

H3CO

1 2

Figure 5. (a) PACE of AGP sequentially digested with: 1, a-L-arabinofuranosidase (x2 di(x2 digestion), exo-b-(1?3)-galactanase, b-glucuronidase and endo-b-(1?6)-galactanasarabinofuranosidase, exo-b-(1?3)-galactanase, b-glucuronidase and endo-b-(1?6)-galacanalysis. (c) MALDI-CID of Gal4Ara oligosaccharide shown in (b).

charide. In particular, the series of 1,5X ions reveal the distributionof the Gal residues on the chain, whilst the 0,4A non-reducing endions confirm this sequence and the (1?6) glycosidic linkagesbetween the Gal residues. The reducing end ions 1,5X3a and 1,5X3b

suggest that the L-Ara residue is linked to the penultimate Gal ofthe oligosaccharide, whilst the presence of the reducing end 0,2X2

and the non-reducing end 3,5A2 ions, reveal that the linkage occursat the C-3 of that Gal residue. Therefore, the oligosaccharide wasidentified as b-Galp-(1?6)-[L-Ara-(1?3)]-b-Galp-(1?6)-b-Galp-(1?6)-b-Galp.

Since the L-Ara could not be removed by a-L-arabinofuranosi-dase digestion, we hypothesised that L-arabinopyranose (L-Arap)

1224.6 1607.4 1990.2Mass (m/z)

1409

.4

1249

.312

05.3

1613

.4

1453

.4

1773

.5

1977

.5

ra

al4Ara

2

Gal5AraGal

5Ara

2

Gal6Ara

Gal6Ara

2

Gal6Ara

3Gal

7Ara

3

722.4 913.2Mass (m/z)

871.

289

9.2

969.

1

693.

2

827.

2

665.

3

795.

2

999.

1

765.

2

575.

3

O

O

O

O

1045

.2

[M+Na]+

1,5X3α

2

3,5A30,4A

3

E3

H3α

D3

1,5X3β

Y3β

Y3α H

4

G4

H3β

E4

D4

(b)

(c)

Y1

Y2

3α

2 1,5X1

3

0,4A3

H2

1,5X2

OCH3

OCH3

OCH3

H3CO

H3CO

H3CO

H3CO

CH2

CH2

gestion), exo-b-(1?3)-galactanase and b-glucuronidase; 2, a-L-arabinofuranosidasee. (b) MALDI-ToF MS spectrum of per-methylated oligosaccharides from the a-L-tanase digestion. The m/z 1045.3 ion marked by a (*) was selected for MALDI-CID

2654 T. Tryfona et al. / Carbohydrate Research 345 (2010) 2648–2656

residues may be present. In order to investigate whether L-Ara waspresent in its pyranose form, the wheat flour AGP was digestedwith a recently identified b-L-arabinopyranosidase.34 This b-L-arabinopyranosidase is able to remove b-(1?3)-linked L-Arapresidues from AGPs. Indeed, digestion with this b-L-arabinopyra-nosidase after a-L-arabinofuranosidase, followed by hydrolysiswith the exo-b-(1?3)- and endo-b-(1?6)-galactanases led toincreased hydrolysis of the chains. This was evident from the in-creased intensity of shorter oligosaccharides and decreased inten-sity of very long oligosaccharides (Fig. 6, compare lane 1 and lane2). However, mass spectrometric analysis indicated that galacto-oligosaccharides plus L-Ara were still present (data not shown)even after exhaustive enzymatic digestion as above. We hypothe-sised that the L-Arap residues might obstruct the removal of allL-Araf by the a-L-arabinofuranosidase. Should this hypothesis becorrect, further digestion of the b-L-arabinopyranosidase treatedsample shown in lane 2 with a-L-arabinofuranosidase and endo-b-(1?6)-galactanase should hydrolyse the long oligosaccharideladder to release Gal and galactobiose. As a control, the samplein lane 1 (lacking the b-L-arabinopyranosidase treatment) was alsosubjected to the above enzyme digestions. Lanes 3 and 4 (Fig. 6)show that essentially all the substitutions of the b-(1?6)-galactanare removed by the combination of a-L-arabinofuranosidase,b-L-arabinopyranosidase and b-glucuronidase when used in theappropriate order. We can therefore conclude that L-Arap residuesare indeed present on wheat AGP. Some of these are terminal andmay protect an L-Araf residue from a-L-arabinofuranosidase diges-tion, whilst others are sensitive to the b-L-arabinopyranosidaseonly after the a-L-arabinofuranosidase treatment, and thereforemay be present in short side chains, for example in the structures

1 2 3 4

Ara

Gal

(Gal)3

(Gal)2

(Gal)4

Figure 6. Arap substitutions revealed by PACE. AGP sequentially digested with: 1, a-L-arabinofuranosidase (x2 digestion), exo-b-(1?3)-galactanase, b-glucuronidase andendo-b-(1?6)-galactanase; 2, a-L-arabinofuranosidase (x2 digestion), b-L-arabinopyr-anosidase, exo-b-(1?3)-galactanase, b-glucuronidase and endo-b-(1?6)-galactanase;3, a-L-arabinofuranosidase (x2 digestion), b-L-arabinopyranosidase, exo-b-(1?3)-galactanase, b-glucuronidase, endo-b-(1?6)-galactanase, a-L-arabinofuranosidaseand endo-b-(1?6)-galactanase (x2 digestion); 4, a-L-arabinofuranosidase (x2 diges-tion), exo-b-(1?3)-galactanase, b-glucuronidase, endo-b-(1?6)-galactanase, a-L-arab-inofuranosidase and endo-b-(1?6)-galactanase (x2 digestion).

b-Arap-(1?3)-a-Fraf-(1?3)-Galp or a-Fraf-(1??)-b-Arap-(1?3)-a-Fraf-(1?3)-Galp.

2.4. Proposed model of the average structure for thecarbohydrate moieties of wheat flour AGP

Combining all data from the enzyme digestions, PACE and MSwe propose a model for the average AG of wheat flour AGP(Fig. 7). The model revises the current view of the structure7,14–16

in several ways. First, we have demonstrated that it contains a b-(1?3)-galactan backbone. Second, highly variable length b-(1?6)-galactan side chains branch from this backbone. Third, weshowed that the side chains can possess GlcA at their non-reducingtermini. Unexpectedly, the AG also contains L-Arap residues on theb-(1?6)-galactan side chains. The work has emphasised the enor-mous heterogeneity in the structure of the wheat flour AGP.

Wheat flour AGP contains three Hyp residues modified by an AGchain.7 Since the precise molecular weight of wheat flour AGP isstill unclear and can range from 22 kDa up to 70 kDa,7,12,14,15

depending on the measuring method used,16 average molecularweight of 20–68 kDa for the carbohydrate component of the AGPcan be calculated. This corresponds to about 30–100 Gal and 15–50 L-Ara residues, per AG chain. Based on our PACE and MALDI-ToF-MS data, most of the b-(1?6)-galactan side chains are longerthan DP 5 and can reach a DP of 20 or more. Therefore, a few longb-(1?6)-galactan chains can be present in each AG, and the b-(1?3)-galactan core could be relatively short. In contrast to thelong side chains found here, most characterised b-(1?6)-galactanside chains in AGs are short, of only one or two residues.1,8,17,28,33

Recent linkage analysis and 13C NMR studies of a 125 kDa AGP iso-lated from whole grain of wheat by b-glucosyl Yariv precipitationsuggest that the carbohydrate moiety consisted of a b-(1?3)-galactan backbone, linked in C-6 to single Gal residues.35 On theother hand, using AG-specific enzymes, long b-(1?6)-galactanshave been reported in radish root AGP.21 Divergent structures ofAGs have been reported for the mugwort (Artemisia vulgaris) pollenallergen Art v 1, which contains branched arabinan side chainslinked in C-3 of a b-(1?6)-galactan core.36

Our data are consistent with the earlier model of wheat flourAGP in that the long b-(1?6)-galactan chains are heavily substi-tuted with L-Araf residues at C-3.12 We found no evidence for thesubstitution of the b-(1?6)-linked galactan side chains with a sin-gle b-Gal residue as previously suggested.16 We have now shownthat single GlcA residues are also present, located at the non-reducing termini of some short b-(1?6)-galactan chains. This isin accordance with reports on type II AG side chain substitutionsfrom many plants, but not reported previously from wheat flourAGP.1,5 In addition, we found evidence that the b-(1?6)-galactanside chains are also substituted with L-Arap residues. Terminal L-Arap has been reported on short branches of b-(1?6)-galactanfrom Mongolian larchwood (Larix dahurica L.)37 and gum Arabic(Acacia senegal),5 for example. L-Arap residues of rhamnogalacturo-nan I (RG-I) of soybean38 and mung bean (Vigna radiata) hypocot-yls39 may act as a signal for termination of elongation of thegalactan chain.40 We consider that some of the L-Arap of wheatflour AG may be terminal, and other L-Arap is substituted with L-Araf residues, based on the need for sequential enzyme digests toremove both sugars. To our knowledge this is the first time thatthe presence of L-Arap on wheat flour AGP has been reported.

In conclusion, our work shows that the wheat flour AG struc-ture has more similarity to some other AGs than previouslythought. It is unclear whether the structures found here are trulydifferent from other AGPs, or whether the use of the enzymes,PACE and MS has revealed a clearer picture. We are currentlyapplying these methods to AGPs from other species to investigatethis question.

Figure 7. A proposed model of one of the many possible structures of the carbohydrate component of the Cadenza wheat flour AGP, and its hydrolytic digestion by variousenzymes. The glycosidic linkages cleaved by each AG-specific enzyme are indicated by: , a-L-arabinofuranosidase; , b-L-arabinopyranosidase; , exo-b-(1?3)-galactanase;

, endo-b-(1?6)-galactanase; , b-glucuronidase.

T. Tryfona et al. / Carbohydrate Research 345 (2010) 2648–2656 2655

3. Experimental

3.1. AG specific enzymes and preparation of wheat flour AGP

a-L-Arabinofuranosidase (EC 3.2.1.55), exo-b-(1?3)-galactanase(EC 3.2.1.145), b-glucuronidase (EC 3.2.1.31), endo-b-(1?6)-galactanase (EC 3.2.1.164) and b-L-arabinopyranosidase were pre-pared by methods described previously.19–21,27,31,32

Cadenza wheat flour water extractable AGP was isolated by themethods of Loosveld et al.41 and van den Bulck et al.16 except thatAX digestions were carried out with recombinant xylanase(NpXyl11B, a gift from Prof Harry Gilbert). Cadenza wheat AGPpreparations were freeze dried and kept at �20 �C.

3.2. TFA hydrolysis and HPAEC-PAD monosaccharide analysis

Samples were hydrolysed in 2 M trifluoroacetic acid (TFA) for1 h at 120 �C. TFA was removed by evaporation under vacuum.Monosaccharide composition of the TFA hydrolysed material wasperformed on a PA20 chromatography column (Dionex, CA, USA)using protocols adapted from Øbro et al.42 In particular, the elutionprogramme consisted of an initial isocratic elution in 2 mM NaOHfrom 0 to 20 min, followed by a linear gradient up to 800 mM

NaOH from 20 to 40 min, followed by an isocratic wash step in200 mM NaOH from 40 to 50 min. The column was then equili-brated for 10 min in 2 mM NaOH.

3.3. Enzymatic hydrolysis and PACE analysis

AGP (1 mg) was digested with approx. 16 mU enzyme at37 �C for 24 h. If a second enzyme digestion followed, the firstenzyme was inactivated for 5 min at 100 �C and the samplewas dried in a rotary evaporator, before dissolving in the appro-priate new buffer. The buffer composition was as follows:50 mM ammonium acetate buffer (pH 5.0, 100 lL); 20 mMammonium acetate, pH 4.6; 10 mM ammonium acetate pH 4.3containing 50 mM KCl; 20 mM ammonium acetate pH 3.5 con-taining 150 mM NaCl, and 0.2 M ammonium acetate buffer pH4.0, for a-L-arabinofuranosidase, exo-b-(1?3)-galactanase, b-glucuronidase, endo-b-(1?6)-galactanase and b-L-arabinopyra-nosidase, respectively.

For PACE, the derivatization of carbohydrates with the fluoro-phore 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) wasperformed according to previously developed protocols.27 Carbo-hydrate electrophoresis and PACE gel scanning were performedas described by Goubet et al.27,43

2656 T. Tryfona et al. / Carbohydrate Research 345 (2010) 2648–2656

3.4. AG oligosaccharide sample desalting and clean-up

Following the enzyme digestions and prior to perdeutero-methylation or per-methylation, released peptides and enzymeswere removed using reverse-phase Sep-Pak C18 cartridges (Waters)as follows: prior to use, the columns were washed with 5 mL 99.8%MeOH (HPLC grade), followed by 5 mL of 5% (v/v) acetic acid (Fish-er). The samples were dissolved in 0.5 mL of 5% (v/v) acetic acidand were loaded onto the cartridge. The AG oligosaccharides wereeluted with 5 mL of 5% (v/v) acetic acid and were lyophilised. Drysamples were dissolved in 0.5 mL of 5% (v/v) acetic acid and weredesalted using 2 mL Dowex beads (50 � 8, H+ form, 50–100 mesh)equilibrated in 5% acetic acid. AG oligosaccharides were elutedwith 4 mL of 5% (v/v) acetic acid. Finally, purified samples werelyophilised.

3.5. Per- and perdeutero-methylation of AG oligosaccharides

Per-methylation of glycans was performed using the NaOH slur-ry method described by Ciucanu and Kerek44 using 1 mL of methyliodide (Fluka) or deuteromethyl iodide in the case of perdeutero-methylation. Dry samples were resuspended in 100 lL MeOHand were kept at room temperature for MALDI-ToF-MS analysis.

3.6. MALDI-ToF/ToF-MS/MS

Methylated samples (5 lL) dissolved in MeOH were mixed with5 lL of 2,5-DHB matrix (10 mg/mL dissolved in 50% MeOH) and1 lL of the mixture was spotted on a MALDI target plate and ana-lysed by MALDI-ToF/ToF-MS/MS (4700 proteomics analyser, Ap-plied Biosystems, Foster City, CA, USA) as previously described.25

High energy MALDI-CID spectra were acquired with average10,000 laser shots/spectrum, using a high collision energy (1 kV).The oligosaccharide ions were allowed to collide in the CID cellwith argon at a pressure of 2 � 10�6 Torr.

3.7. b-Galactosyl Yariv diffusion assay

A Yariv diffusion assay for the AGP was performed using a pro-tocol adapted from van Holst and Clarke.45 Wheat flour AGP sam-ples were dissolved in 1 mL of 0.15 M NaCl buffer solutioncontaining 0.02% sodium azide. Agarose gels (1%, approximately3 mm thick) containing the b-galactosyl Yariv reagent (0.002%),0.15 M NaCl and 0.02% sodium azide were poured in petri dishes.Polysaccharide solution (15 lL) was pipetted into wells in thegel. The buffer without the polysaccharides was used as blank,and gum Arabic (Sigma) and oat spelt xylan (Sigma) as positiveand negative controls, respectively. The petri dishes were sealedand incubated in the dark at ambient temperature for 2 days to al-low the halo to develop.

Acknowledgements

The project was supported by the Food Standards Agency UK,Grant G03029. We thank Harry Gilbert for the gift of the xylanase.

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