Phosphate esters in amylopectin clusters of potato tuber starch

International Journal of Biological Macromolecules 48 (2011) 639–649

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Phosphate esters in amylopectin clusters of potato tuber starch Jeanette Wikman a,∗ , Flemming Hofmann Larsen b , Mohammed Saddik Motawia c , Andreas Blennow c , Eric Bertoft d,1 a

Department of Biosciences, Åbo Akademi University, Artillerigatan 6, FI-20520 Turku, Finland Department of Food Science, Quality & Technology, University of Copenhagen, 1958 Frederiksberg C, Denmark c VKR Centre of Excellence Pro Active Plants, Department of Plant Biology, Faculty of Life Sciences, University of Copenhagen, 1870 Frederiksberg C, Denmark d Department of Food Science, Swedish University of Agricultural Sciences, P.O. Box 7051, S-75007 Uppsala, Sweden b

a r t i c l e

i n f o

Article history: Received 3 January 2011 Received in revised form 7 February 2011 Accepted 8 February 2011 Available online 16 February 2011 Keywords: Starch structure Amylopectin cluster Starch phosphorylation Antisense suppression

a b s t r a c t Starch phosphate is important in starch metabolism and in order to deduce its location and structural effects in clusters and building blocks of amylopectin, these were isolated from a normal potato (WT) and two starches with antisense suppressed glucan water dikinase (asGWD) activity and starch branching enzyme (asSBE) activity possessing suppressed and increased phosphate contents, respectively. Neutral N-chains and phosphorylated P-chains of the amylopectin macromolecules were similar in WT and asGWD, whereas asSBE possessed considerably longer P-chains. Cluster ␤-limit dextrins were isolated by ␣-amylase treatment and successive ␤-amylolysis. Cluster sizes were generally smaller in asSBE. The building block composition of neutral N-clusters were very similar in WT and asGWD, while asSBE was different, containing less blocks with degree of polymerization (DP) > 14. Phosphate content of the Pclusters of WT and asGWD was rather similar, while asSBE contained highly phosphorylated P-clusters with proportionally more P-chains and a low degree of branching. The average chain lengths of the Pclusters were, however, similar in all samples. Our data demonstrate only minor effect on the cluster structure in relation to phosphate deposition suggesting conserved reaction patterns of starch phosphorylation. Models are suggested to account for the principle structural and functional effects of starch phosphate esters. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Amylopectin is the major macromolecule of starch. It is a branched higher-molecular-mass ␣-(1→4)-glucan with frequent ␣-(1→6)-glucan branch points. The linear side chains are divided into short A-chains that are not carrying other chains and short B1chains that are carrying other chains (A- and/or B-chains) through ␣-(1→6)-linkages. The molecular association of the amylopectin chains is complex. Chromatographic data based on enzymeassisted degradation suggests that the short chains are clustered and interlinked through long B2- and B3-chains. Combined with Xray and electron microscopic data it is suggested that their external parts form parallel double helices that pack in a side by side manner to form different crystalline arrays found in the native starch granules [1,2]. Small angle X-ray information indicates that the double helices form approximately 5–6 nm thick crystalline lamellae that alternate with 3–4 nm thick amorphous lamellae where most of

∗ Corresponding author. Tel.: +358 50 5208 604: fax: +358 22 410 014. E-mail address: jeanette.wikman@abo.fi (J. Wikman). 1 Present address: Food Science Department, University of Guelph, Guelph, ON N1G 2 W1, Canada. 0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.02.005

the branches are found [3]. Whereas the crystalline lamellae are relatively easy to analyse using solid state technologies and reliable data quite well describe these structures, the organization of the amorphous branched lamellae is virtually unknown [4]. Molecular models suggest that the branch point seems to be specifically stabilized by a structured water molecule [5] and positioning of the branch points along the ␣-(1→4) backbone can direct the registration of double helices to align into the crystalline lattices [6]. However, experimental data are lacking to provide evidence for these models. Most starch types contain phosphate monoesters [7], which are mainly found in the amylopectin component [8]. Phosphate groups provide the starch granule with highly hydrated local cavities important for amorphisation and industrially, phosphate esters are important to increase starch paste clarity and viscosity [9–12]. In the plant cell, starch-phosphate esters are necessary for complete metabolism of the starch granules and they stimulate degradation of the starch, possibly by introducing local hydration within the crystalline lamellae [4]. A majority of the phosphate monoesters appears to be located on the B-chain near the centre of the unit chain and more than 9 glucosyl residues from the branch point [8]. If the phosphate monoesters are closer to the branch points, phosphate located at C-6 can be found on glucose units as close as

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one residue from the branch point, while phosphate located at C-3 can be found on glucose units that are approximately two residues away from the branch point [13]. The degree of phosphorylation correlates with the chain length (CL) and crystalline polymorph of amylopectin [3,14], which points at an interesting relationship between crystalline polymorphic structures and their amorphisation required for degradation [4]. However, the distribution of the phosphate monoesters in relation to branched clusters is not known. Phosphate esters are located in crystalline starch but substantial amounts are found in amorphous starch [1,15]. Hence, the preferential presence of phosphate in moderately long amylopectin chains to be suggested to participate in crystalline segments and its simultaneous presence in amorphous regions provides an enigma and requires focused investigations. Amylopectin is produced at the surface of the granule by mainly two enzymes, soluble starch synthase (SSS) and starch branching enzyme (SBE). SSS elongates the ␣-(1→4)-chain at the non-reducing end, as catalyzed by a transfer reaction involving the sugar donor ADP-glucose. The formation of the ␣-(1→6)-linkages is catalyzed by SBE by a transfer reaction of the existing elongated chains [16]. Debranching enzyme activity has been suggested to play a crucial role for the production of the crystalline lamellae [17]. The enzyme responsible for the phosphorylation of amylopectin was found to be ␣-glucan, water dikinase (GWD) [4,10,18]. GWD is mainly responsible for phosphorylation at the C-6 position, while the homologue GWD3 (or phosphoglucan, water dikinase, PWD) phosphorylates nearby C-6 phosphorylated chains at the C-3 position [19–21]. As a result, about 30% of the phosphate monoesters in amylopectin are located at C-3 [14,22]. Even though the enzyme activities involved in starch biosynthesis are well known, their interplay is enigmatic and so far starch granules cannot be produced in vitro. To date, the most efficient way of getting detailed molecular information on the amorphous branched regions of the starch granule is enzyme-assisted degradation combined with chromatographic analysis of the generated products. In previous studies we have employed partial enzymatic hydrolysis using ␣-amylase, ␤amylase and phosphorylase to deduce cluster and their building block structures. Using ␣-amylase from Bacillus amyloliquefaciens, fractions containing clusters of short chains were obtained from amylose-free potato starch (PAPS) [23]. By treating these fractions with exo-acting enzymes (phosphorylase a and ␤-amylase) the external chains were reduced into short remnants containing only 1–2 residues. The internal parts of the cluster, including all the branches as well as the internal chain segments, were left in the remaining limit dextrins. These were then further subjected to a second hydrolysis by the ␣-amylase to produce near-limit dextrins, named building blocks, in order to analyse the molecular organization of the inner structure [24]. In these studies a cluster was defined as a group of chains, in which the branches are found closer than ∼9 residues from each other, whereas building blocks were characterized as densely branched areas, in which the average chain length between branches was only ∼2 glucosyl residues. Inside clusters the building blocks are interconnected by inter-block segments that have an apparent length of 7–8 glucosyl residues [23,24]. Clusters in PAPS are of rather homogenous size in the DP-range 50–70 and contain 7–11 chains. Differences in the degree of branching indicated that there were differences in the fine structure among the clusters [23]. The aim of the present investigation was to study the distribution of C-3 and C-6 phosphate monoesters among the clusters of potato amylopectin. The starches included starch prepared from a normal potato of the cultivar Dianella, and two modified starches, in which the amylose and phosphate levels were engineered by antisense technology. These included an antisense suppressed GWD [19,25] having low phosphate level and slightly elevated amy-

lose content, and antisense suppressed SBE [25,26] also possessing slightly elevated amylose, but in addition highly elongated amylopectin chains and a very high phosphate level. Amylopectin from these starches were (a) hydrolyzed by ␤-amylase to generate ␤-limit dextrins of the amylopectin and (b) hydrolyzed by ␣-amylolysis until the hydrolysis rate was insignificant generating single clusters [27]. The isolated clusters where then further hydrolyzed by ␤-amylolysis to receive the ␤-limit dextrins of the clusters. ␤-Limit dextrins of the amylopectin were analysed for their internal chain composition and the clusters where analysed for their phosphate content and internal chain composition. In spite of the profound effect of starch phosphate in starch metabolism, the data suggest that the phosphate groups have only minor influence on the unit chain composition of clusters and that the differences are mainly found in the internal chain structure of the amylopectin. 2. Materials and methods 2.1. Starch sample and enzymes Starch was extracted from Dianella wild type (WT), Dianella antisense GWD (asGWD) and Dianella antisense SBE (asSBE) as described [26,28]. Bacillus subtilis ␣-amylase, liquefying type (also known as B. amyloliquefaciens ␣-amylase, EC 3.2.1.1) with an activity of 1000 U/mg solid at pH 6.5 and 25 ◦ C was from Seikagaku Corporation (Tokyo, Japan). Barley ␤-amylase (EC 3.2.1.2), specific activity ca. 1400 U/mg, Pseudomonas isoamylase (EC 3.2.1.68), ca. 280 U/mg, and Klebsiella planticola pullulanase (EC 3.2.1.41), 42 U/mg, were from Megazyme International (Wicklow, Ireland). 2.2. Amylose and starch-phosphate analysis Amylose was determined by two different methods: iodine colorimetry using the method described [29] and debranching method described by Sargeant [30] with minor modifications: 5 mg starch was dissolved in 2 mL of 0.1 M NaOAc buffer (pH 3.5) on a boiling water bath. The sample was then debranched by isoamylase (0.6 ␮L/mg) and the sample was stirred overnight at 35 ◦ C and the reaction was terminated by adding 5 M NaOH (25 ␮L/mL). The debranched sample was analysed by gel-permeation chromatography (GPC) as described below. The amylose content was calculated as the relative area of the distinct peak of long chains. Starch bound phosphate was determined using the method described by Bay-Smidt et al. [31]. 2.3. Isolation of amylopectin The amylopectin was isolated from native starch by precipitation in 1-butanol and isoamyl alcohol essentially according to Klucinec and Thompson [32] with the following modifications. Granular starch (2 g) was dispersed in 40 mL 90% (v/v) dimethyl sulfoxide (DMSO) by heating in a water bath at 90 ◦ C with constant stirring for 3 h under a stream of nitrogen gas. The sample was slowly cooled to 65 ◦ C before 4 volumes of ethanol was added dropwise using a peristaltic pump. The mixture was allowed to cool to room temperature and then centrifuged at 8000 × g and 4 ◦ C for 60 min. The supernatant was discarded and the pellet was washed once in ethanol. The non-granular starch was then stirred in 90% DMSO (56 mL, 90 ◦ C) under nitrogen until completely dissolved. A mixture of 23.5 mL 1-butanol and 23.5 mL isoamyl alcohol in 324 mL H2 O was then dropwise added with a peristaltic pump (∼5 mL/min) at 80 ◦ C using continuous stirring. The mixture was transferred to a preheated insulated box and allowed to slowly cool down to 25–30 ◦ C for 18 h.

J. Wikman et al. / International Journal of Biological Macromolecules 48 (2011) 639–649

641

Amylopectin (AP)

Debranching enzyme

Unit chains β-Amylase

β-limit dextrins

α-Amylase

Debranching enzymes

α-dextrins

dextrin β-limit β li it d t i chains

β -Amylase β-limit dextrins

ION-EXCHANGE CHROMATOGRAPHY

Phosphorylated p y chains (P-chains)

Neutral chains (N-chains)

ION-EXCHANGE CHROMATOGRAPHY

Neutral clusters (N-clusters) α-Amylase

Building Blocks

Phosphorylated clusters (P-clusters) Debranching enzymes

Debranching enzymes

P-cluster chains

N-cluster chains

ION-EXCHANGE CHROMATOGRAPHY

Neutral chains (N-chains)

Phosphorylated chains (P-chains)

Fig. 1. Scheme of amylopectin enzyme-assisted fragmentation and analysis used in this study.

After cooling, the mixture was centrifuged as above and the supernatant, containing the amylopectin fraction, was concentrated under vacuum to 50 mL by rotary evaporation at 50 ◦ C. Three volumes of methanol was added and the sample was left at room temperature overnight. The precipitate was collected by centrifugation, dissolved in hot water (approximately 20 mL) and re-precipitated in three volumes of ethanol for 1 h at room temperature. After centrifugation as above, the precipitate was re-dissolved in hot water, the volume reduced by rotary evaporation and the amylopectin preparation freeze-dried. The amylopectin was then analysed according to the scheme in Fig. 1 and described below.

mixture incubated overnight at room temperature and the reaction stopped on a boiling water bath. The chain lengths were analysed by gel-permeation chromatography (GPC) as described below. 2.6. Anion-exchange chromatography The debranched ␤-LDs (14 mg/mL) were applied on a column (1 cm × 10 cm) of DEAE–Sepharose (Pharmacia, Sweden). The material was collected with a fraction collector (2 mL/fraction). Neutral chains were eluted with deionized water (22 fractions) and phosphorylated chains were eluted with 0.2 M NaCl (20 fractions). The pooled fractions were freeze-dried.

2.4. Production of amylopectin ˇ-limit dextrins 2.7. Isolation of clusters Amylopectin (100 mg) was dissolved in deionized water (48.5 mL) on a boiling water bath and 0.025 volumes of 0.01 M NaOAc buffer, pH 6.0, was added. A solution of ␤-amylase (4 U/mg starch) was added and the reaction mixture incubated overnight at room temperature. The reaction was stopped by boiling and generated maltose was removed using tangential flow filtration (TFF) in a Minimate TFF Capsule containing Omega 10 kDa membrane (Pall Life Sciences, Ann Arbor, MI USA). The carbohydrate concentration was adjusted to 2 mg/mL, the ␤-amylolysis was repeated and the maltose was removed as above. The final ␤-limit dextrins (␤-LDs) were lyophilized. 2.5. Debranching of amylopectin and their ˇ-limit dextrins Amylopectins and ␤-LDs were dissolved in deionized water (7 mg/mL) on a boiling water bath. 0.05 volumes of 0.1 M NaOAc buffer, pH 5.5 and debranching enzymes (isoamylase 0.25 ␮L/mg ␤-LDs and pullulanase 0.25 ␮L/mg ␤-LDs) were added, the reaction

The ␣-dextrins were prepared by ␣-amylase as described by Bertoft [27,33] with minor modifications. The amylopectin preparations (500 mg) were dissolved in 90% DMSO (10 mL) by stirring at room temperature for 4 days. The solution was then diluted with hot deionized water (35 mL) and a solution of ␣-amylase (5 mL, 0.09 U/mL) in 0.01 M NaOAc buffer, pH 6.5, was added. The reaction took place at 25 ◦ C and was terminated after 70 min by adjusting pH to 13 with 5 M NaOH and left for 1 h. Methanol (5 volumes) was then added to precipitate the ␣-dextrins and left for 1 h at room temperature. The precipitate was recovered by centrifugation (1800 × g, 10 min), washed with methanol (ca. 100 mL), for second centrifugation. The precipitate was dissolved in hot deionized water whereafter excess methanol was removed and the sample was concentrated by evaporation (Büchi Rotavapor R-3000). The concentrated ␣-dextrins (5 mg/mL) were then treated with ␤-amylase according to the procedure described elsewhere [34] and the maltose was removed from the sample by gel-permeation

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Table 1 Amylose content in the native starches and amylopectin composition.

9

WT

asGWD

asSBE

Whole starch Am%a Whole starch Am%b AP fraction Am%b CLc ECLd ICLe ␤-Limit value (%)f

20 20 5.4 21.4 13.5 6.9 53.7

31 25 2.5 21.8 13.9 6.9 54.4

39 24 1.9 37.0 20.0 16.0 48.6

a b c d e f

Amylose content by iodine colorimetry. Amylose content in starch or purified amylopectin by the debranching method. Chain length of amylopectin. External chain length = CLAp × ␤-limit value (%)/100 + 2. Internal chain length = CL␤-LD − 2 − 1. ␤-Limit value = (CLAp − CL␤-LD )/CLAp × 100.

Table 2 Concentration of phosphate in whole starches and their phosphorylated cluster. Total P (nmol/mg starch) WT Whole starch P-clusters asGWD Whole starch P-clusters asSBE Whole starch P-clusters

P6 (nmol/mg starch)

P3 (nmol/mg starch)

P6/P3

21.2 115.4

15.2 87.6

6.0 27.8

2.5 3.2

3.1 148.2

2.2 87.6

0.9 60.6

2.4 1.4

73.8 230.2

52.0 182.5

21.8 47.7

2.4 3.8

(a)

AM

AP

6

3

Carbohydrates (relative concentration)

Parameter

0 9

(b)

6

3

0 9

(c)

6

3

0 41

61

81

101

121

Fraction number

2.8. Analysis of cluster structure 2.8.1. Starch-bound phosphate Starch bound phosphate was determined by 31 P NMR. P-clusters (10 mg) were dissolved in 110 mM NaCitrate, pH 4.5 (540 mL) and D2 O (60 mL, 99.0% deuterated and with 5.8 mM TSP-d4). pH was adjusted to 4.5 with 2 M NaOH or 2 M HCl and the solution was transferred to a 5 mm NMR tube. 1 H NMR spectra were recorded on a Bruker Avance III 600 NMR spectrometer operating at 600.13 MHz for 1 H, while 31 P NMR spectra were recorded on a Bruker Avance 400 NMR spectrometer operating at 161.98 MHz for 31 P. Standard maltose 6-phosphate and maltose 3-phosphate were chemically synthesized as described [36] and Table 3 Composition of ␤-LD of amylopectin. Parameter

WT

asGWD

asSBE

CL N-chains by weight (%) N-chains by number (%) N-chains CL P-chains by weight (%) P-chains by number (%) P-chains CL

9.8 85.8 91.8 9.2 13.9 8.2 16.6

11.0 95.0 96.9 11.0 4.3 3.1 15.5

21.7 33.8 67.4 9.8 65.6 32.6 39.3

Fig. 2. Sepharose CL-6B GPC of debranched whole starch (䊉) and purified amylopectin fraction () from (a) WT, (b) asGWD, and (c) asSBE. (AM) and (AP) denote amylose and amylopectin, respectively.

phosphate concentrations determined from P ester specific signal intensity.

2.8.2. Building blocks of N-clusters The N-clusters of the ␤-limit dextrins were treated with ␣amylase to produce building blocks according to Bertoft [24]. The size-distribution of the building blocks was analysed by GPC as described below.

200

9

Carbohydrate (relative concentration)

on two columns of Sephadex-25 (PD-10 columns, Pharmacia, Sweden) coupled in series [35]. The eluate was concentrated to 4 mg/mL by evaporation. The ␤-amylolysis was repeated, the maltose removed, and the sample concentrated as above. The samples were enzymatically treated and analysed according to the scheme in Fig. 1. The samples were also separated on the DEAE anion-exchange chromatography column into neutral clusters (N-clusters) and phosphorylated clusters (P-clusters) as described above and then freeze-dried.

100

60

40

20

BL

10

2

B1

DP A

6

3

0 0

0.3

0.6

0.9

Kav Fig. 3. Superdex 75 GPC of debranched ␤-limit dextrins of WT (), asGWD (䊉), and asSBE (). (A) corresponds to A-chains, (B1) to short B1-chains, and (BL) to long B-chains.

J. Wikman et al. / International Journal of Biological Macromolecules 48 (2011) 639–649

100 9

(a)

60

40 B L

20

2

10 B 1

DP

A

6

Carbohydrate (relative concentration)

3 0 9

(b)

6 3 0 9

(c)

643

The columns were calibrated for branched dextrins of known DP by the method described by Bertoft and Spoof [33]. For the debranched dextrins the column of Superdex 75 was calibrated by determining the reducing carbohydrates with disodium 2,2 bicinchonitate (BCA) reagent. 20 mg amylose-free potato starch (PAPS) was dissolved in deionized water (30 mg/mL) and 0.5 volumes of 0.1 M NaOAc, pH 3.5, was added. The sample was debranched by isoamylase (0.5 ␮L/mg starch), stirred overnight at room temperature and the reaction terminated by boiling. The debranched PAPS (0.8 mL) was applied on the column of Superdex 75 and eluted as above. The pH in the eluted fractions was adjusted to pH 10 by adding 0.04 M NaOH. An aliquot of the fractions (0.1 mL) was diluted with deionized water (0.4 mL) and analysed for total carbohydrate content with the phenol–H2 SO4 reagent [37]. Another aliquot of the fractions (0.5 mL) was measured for the reducing carbohydrates with the BCA reagent according to Doner and Irwin [38] with minor modifications: 0.5 mL of the reagent was added to each fraction and the samples were heated in boiling water for 20 min and then cooled down on ice for 20 min. The absorbance was measured at 560 nm against a blank containing no carbohydrates and a standard sample containing 2 ␮g glucose/mL was used for calibration. DP was calculated as total carbohydrates/reducing ends. 3. Results 3.1. Analysis of whole starch and amylopectin

6 3 0 0

0.3

0.6 Kav

0.9

Fig. 4. Superdex 75 GPC of N- () and P-chains () of ␤-limit dextrins of (a) WT, (b) asGWD, and (c) asSBE. (A) corresponds to A-chains, (B1) to short B1-chains, and (BL) to long B-chains.

2.8.3. Unit chains The material to be debranched was dissolved in deionized water (7 mg/mL) and 0.05 volumes of 0.1 M NaOAc, pH 5.5 was added. The sample was then treated with isoamylase (∼0.3 ␮L/mg) and pullulanase (∼0.3 ␮L/mg), stirred overnight at room temperature and the reaction terminated by boiling. 2.8.4. Anion-exchange chromatography of debranched P-clusters Debranched P-clusters were applied on the DEAE anionexchange chromatography column and separated into N- and P-chains as described above. 2.8.5. Molecular weight distribution Samples of debranched starch were applied to a column (1 cm × 90 cm) of Sepharose CL-6B (Pharmacia, Sweden). The column was eluted with 0. 5 M NaOH at 0.5 mL/min. Samples of dextrin (after ␣-amylolysis, ␤-amylolysis and debranching) were diluted to ∼3 mg/mL and (0.2 mL) applied to a column (1 cm × 90 cm) of Superdex 75 (Pharmacia, Sweden). Samples of building blocks were applied to a column (1 cm × 90 cm) of Superdex 30. The columns were eluted with 0.05 M NaCl at 0.5 mL/min. Fractions (0.5 mL) were collected and their carbohydrate content was analysed with the phenol–H2 SO4 reagent [37].

Proper quantification of amylose is inherently problematic [39,40] and hence, the amylose content of the whole starch was determined by two independent methods: iodine colorimetry and debranching method. The two methods gave different values (Table 1). The values for amylose content of WT was the same for both methods (20%), but for the transgenetically modified starches the debranching method gave significantly lower values (asGWD 25% and asSBE 24%) than the iodine calorimetry method (asGWD 31% and asSBE 39%). It is well known that a modified amylopectin structure, in which the chains are longer than in normal starch, tends to bind more iodine thus resulting in overestimated amylose values [41]. WT starch contained 21.2 nmol phosphate/mg while asGWD contained only 3.1 nmol phosphate/mg starch. In asSBE the content was elevated to 73.8 nmol phosphate/mg starch (Table 2). The ratio of P6 vs. P3 was similar (2.4–2.5) in all starches. In order to analyse the amylopectin chain length profiles, the amylopectin fractions were separated from the native starches by removing the amylose through precipitation in 1-butanol and isoamyl alcohol. The isolated amylopectin fractions that remained in the supernatants contained only small amounts of amylose (between 1.9 and 5.4%) as indicated in the chromatograms (Fig. 2) as minor peaks in fractions 43–51. The chain lengths (CL) of the amylopectin fractions were similar for WT and asGWD (CL ∼21) but longer for asSBE (CL 37). This was seen in the chromatograms as a comparatively broad peak for asSBE (Fig. 2). The external chain length (ECL), the internal chain length (ICL), as well as the ␤-limit value, all showed the same trend, the values for WT and asGWD were similar, whereas the values for asSBE differed (ECL and ICL was higher and the ␤-limit value was lower) (Table 1). 3.2. Analysis of amylopectin ˇ-LDs The amylopectin fractions were subjected to ␤-amylase to produce ␤-limit dextrins (␤-LDs). When ␤-amylase attacks amylopectin, maltose is cleaved off from the non-reducing ends of the chains. However, the enzyme is not able to attack the ␣-(1→6)-

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200 100 60 40 20 10

Carbohydrate (relative concentration)

8

6

200 100 6040 20 10

DP

(a)

6

200 100 6040 20 10

DP

(b)

6

DP

(c)

6 4

2 0 0

0.3

0.6

0.9

0

0.3

K av

0.6

0

0.9

0.3

0.6

K av

0.9

K av

Fig. 5. Superdex 75 GPC of the ␣-amylolysis products from the whole sample () and of the precipitates obtained in methanol () of (a) WT (b) asGWD, and (c) asSBE.

␣-(1→6)-linkage are resistant to the enzyme [44,45]. Phosphate groups at 3- and 6-position were shown to be resistant to the enzyme in similar way as the branches [43]. The size-distribution of the ␣-dextrins was analysed by GPC and possessed bimodal pattern with two populations (Fig. 5). The major fraction was obtained as a precipitate in methanol, while the minor fraction remained in the supernatant and is known to contain mostly maltooligosaccharide fragments from the external chains of the clusters [23]. The precipitate was further hydrolyzed by ␤amylase to produce ␤-limit dextrins of the clusters. ␤-Amylolysis removed most of the external chains remaining after the ␣-amylase attack. The clusters were applied on an anion-exchanger to separate the material into neutral clusters (N-clusters) and phosphorylated clusters (P-clusters). The yield of the P-clusters was determined by weight as well as by number (Table 4). WT gave a yield of the Pclusters of 29% by weight, corresponding to 24% by number. For asGWD the yield was 7% by weight and 4% by number, whereas the yield from asSBE was much higher: 68% and 59%, respectively. The degree of polymerization (DP) of the N-clusters in WT and asGWD was 30–35, which was smaller than the P-clusters (DP 45–51). In asSBE both the N-clusters (DP 23) and the P-clusters (DP 35) were smaller than in the other samples (Table 4). N-clusters of WT and asGWD showed similar size-distribution profiles, whereas the peaks of both the N-clusters and the P-clusters of asSBE were narrower (Fig. 6).

linkages leaving stubs of 1–3 glucosyl residues on the non-reducing end side. After debranching, these stubs can be identified as Achains with lengths of 2–3 residues. Also, if a phosphate group is bound to the ␣-(1→4)-chain, the enzyme is not able to bypass it [8,42,43]. The chain length profiles of the ␤-LDs analysed by GPC possessed similar CL for WT and asGWD (9.8 and 11.0, respectively), while asSBE had noticeably longer chains (CL 21.7) (Table 3 and Fig. 3). The debranched ␤-LDs were subjected to ion-exchange chromatography to separate neutral chains (N-chains) and phosphorylated chains (P-chains). Only 4.3% by weight of the asGWD material was eluted as P-chains, and this corresponded to 3.1% by number of all chains (Table 3). The amount in WT was higher (13.9% and 8.2%, respectively), whereas asSBE possessed a very high amount of P-chains (65.6% and 32.6%, respectively). The chain length profiles of the N- and P-chains were analysed by GPC. Whereas the profiles of the N-chains for all three samples were similar (CL 9.2–11.0), the profiles of the P-chains showed distinct differences (Fig. 4). The P-chain profiles of WT and asGWD were similar with the peak position at DP 20 and containing mainly short B1- and long B-chains. However, the P-chain profile of asSBE possessed mainly very long B-chains and the peak position was at DP ∼100. The P-chains of WT and asGWD had similar CL values (16.6. and 15.5, respectively). In contrast, the chains of asSBE were considerably longer (39.3) (Table 3). 3.3. Analysis of clusters

3.4. Analysis of N-clusters The amylopectin samples were hydrolyzed by B. subtilis ␣amylase to produce ␣-dextrins. While ␣-amylase readily attacks the longer internal chains of amylopectin, the shorter chains are almost entirely resistant to the hydrolysis. Studies on products of ␣-amylase have shown that one or two linkages next to an

Carbohydrate (relative concentration)

400200 100 6040 9

20 10 6

The N-clusters were further hydrolyzed by ␣-amylase and debranching enzymes. By adding an excess of ␣-amylase, the building block composition of the N-clusters was obtained. The building blocks of asSBE were smaller (DP 8.9) than in WT (DP 11.7) and

400200 100 6040

DP

(a)

20 10 6

400200 100 6040

DP

(b)

20 10 6

(c)

6

3

0 0

0.3

0.6

K av

0.9

0

0.3

0.6

K av

0.9

0

0.3

0.6

K av

Fig. 6. Superdex 75 GPC of N-clusters () and P-clusters () of (a) WT, (b) asGWD, and (c) asSBE.

0.9

DP

J. Wikman et al. / International Journal of Biological Macromolecules 48 (2011) 639–649

645

Table 4 Composition of clusters in potato amylopectin. Parameter

WT

Yield (%) Rel. no (%)a DP CLb ICLc DB (%)d NCe

asGWD

asSBE

N-clusters

P-clusters

N-clusters

P-clusters

N-clusters

P-clusters

71 76 35 5.4 3.0 15.6 6.4

29 24 45 6.4 4.1 13.4 7.1

93 96 30 5.7 3.6 14.2 5.3

7 4 51 6.4 4.0 13.7 8.0

32 41 23 5.7 4.4 13.3 4.1

68 59 35 7.6 6.1 10.3 4.6

a Relative number of N-clusters = ((YieldN-clusters /100)/DPN-clusters )/[((YieldN-clusters /100)/DPN-clusters ) + ((YieldP-clusters /100)/DPP-clusters )]; clusters = ((YieldP-clusters /100)/DPP-clusters )/[((YieldP-clusters /100)/DPP-clusters ) + ((YieldN-clusters /100)/DPN-clusters )]. b Average chain length. c Internal chain length = ((CL − 2) × NC/(NC − 1)) − 1. d Degree of branching = (NC − 1)/DP × 100. e Number of chains = DP/CL.

Table 5 Compositional characterization of building blocks from N-clusters. WT

asGWD

asSBE

DP (Total) Bbla (wt.%) Bbla (mol.%) Linearb (mol.%) DPBbl DPLinear NBbl/clusterc IB-DPd

8.5 88.1 64.4 35.6 11.7 2.9 2.6 1.6

7.0 84.0 53.8 46.2 11.0 2.4 2.3 2.1

5.4 76.3 46.0 54.0 8.9 2.4 2.0 2.8

a

c d

Bbl = branched building blocks. Linear = linear fragments from inter-block segments. Number of building blocks per cluster = Bbl (wt.%)/100 × DPN-cluster /DPBbl . Inter-block DP = linear (mol.%) × DPLinear /Bbl (mol.%).

15

Carbohydrate (relative concentration)

5

4

3

2

number

of

P-

3.5. Analysis of P-clusters

Parameter

b

relative

1

12 9

The phosphate content of the P-clusters was determined by NMR (Table 2). The phosphate was more concentrated in the Pclusters compared to the whole starch. There was more phosphate at the C-6 position than at the C-3 position. The ratio of P6 vs. P3 in the P-clusters was higher in both asSBE and WT than in the whole starches, while asGWD possessed a lower P6/P3 ratio. The CL of the P-clusters was similar in WT and asGWD (6.4), while it was higher (7.6) in asSBE (Table 4). asSBE clusters possessed fewer chains at DP 2–4 and slightly more chains at DP 20–40 (Fig. 8b) than the other samples. The chains were applied on the anion-exchanger to separate Nchains and P-chains. The yield of P-chains was similar for WT (38% by weight, 18% by number) and asGWD (43% and 24%, respectively), while it was higher for asSBE (65% and 45%, respectively) (Table 7). The unit chain profiles of the samples were all rather similar (Fig. 9) and the CL of the N-chains in all three samples was ∼5.5. P-chains were longer with CL ∼13.3. The number of P-chains per cluster was only 1.3 in WT and slightly higher (∼2) in the other samples. The number of N-chains per cluster was ∼6 in WT and asGWD and lower in asSBE (Table 7).

6

4. Discussion 3 0

0

0.3

0.6

0.9

Kav Fig. 7. Superdex 30 GPC of building blocks of N-clusters from WT (), asGWD (䊉), and asSBE (). Numbers 1–5 indicate different groups of building blocks.

asGWD (DP 11.0) (Table 5), while the DP for the linear material was similar (DP ∼2.5). The number of building blocks (NBbl) per cluster was low and ranged from 2.0 to 2.6. According to Bertoft [24], the building blocks can be divided into 5 groups denoted 1–5 (Fig. 7). The comparison of these groups showed that the material had a similar weight-distribution in clusters from WT and asGWD. The distribution in asSBE was different (Table 6) showing less material with DP ≥ 14. It is possible to estimate the inter-block DP (IB-DP) inside the clusters (Table 6) from the formation of linear blocks at DP 1–5, that were formed by the ␣-amylase attack on the internal chains that inter-connected the branched blocks [24]. Debranching of the N-clusters revealed the chain structure (Fig. 8a) and the CL of the N-clusters were similar for all three samples (CL ∼5.7) (Table 4).

In this study antisense suppression technology was used to address specific structural starch alteration as a function of amylopectin cluster chain structure. Antisense of GWD and SBE resulted in specific decrease (asGWD) and increase (asSBE) in phosphate. The longer amylopectin chains generated in the asSBE tuber (Table 1) correlates with an increased level of phosphate (Table 2) as also found in other studies [26,46,47]. However, the P6/P3 ratio was unaffected. Hence, the starch models used in this work provide starches with some specific structural attributes. Inner chain structures of these three model starches were assessed by ␤-amylase leaving 1–3 glucosyl stubs close to the ␣-(1→6)-linkages. These stubs identify the A-chains if they are 2–3 residues. For C-6 linked Table 6 Size-distribution of groups of building blocks in N-clusters. Group

DPa

1 2 3 4 5

<5 5–9 10–13 14–19 >20

a

Weight% WT

asGWD

asSBE

11.9 34.6 22.5 12.7 18.3

16 37.1 20.8 10.2 15.9

23.7 48.9 16.7 5.7 4.9

DP-ranges according to Bertoft [24].

646

J. Wikman et al. / International Journal of Biological Macromolecules 48 (2011) 639–649

100

Carbohydrate (relative concentration)

12

60

40

20

10 6 3

(a)

10

100

DP

60

40

10 6 3

20

DP

(b)

8 6 4 2 0

0

0.3

0.6

0

0.9

0.3

Kav

0.6

0.9

Kav

Fig. 8. Superdex 75 GPC of debranched (a) N-clusters and (b) P-clusters, WT (), asGWD (䊉), and asSBE ().

Table 7 Unit chain composition of P-clusters. Parameter

WT

Yield (%) Rel. no. (%)a CL NC/clusterb

asGWD

asSBE

N-chains

P-chains

N-chains

P-chains

N-chains

P-chains

62 82 5.0 5.7

38 18 13.5 1.3

56 76 5.5 6.1

43 24 13.0 2.0

35 55 5.7 2.6

65 45 13.3 2.1

a Relative number of N-chains = ((YieldN-chains /100)/CLN-chains )/[((YieldN-chains /100)/CLN-chains ) + ((YieldP-chains /100)/CLP-chains )]; chains = ((YieldP-chains /100)/CLP-chains )/[((YieldP-chains /100)/CLP-chains ) + ((YieldN-chains /100)/CLN-chains )]. b Number of N-chains per cluster and number of P-chains per cluster, respectively, calculated as Rel. no. (%)/100 × DPP-cluster /CLP-cluster .

phosphate groups the enzyme leaves one or no glucose residue. However, if the phosphate group is at the C-3 position the stub length is not known [8,42,43]. Hence, A-chain phosphate groups cannot be readily identified. Our data show that the N-chains of all three starch models were similar showing that all the samples had similar A-chain and internal B-chain lengths. However, the P-chain population possessed very different size-distributions indicating differences in phosphate distribution along the chains independent of the inner chain segments. The P-chains of WT and asGWD contained mainly a mixture of long and short B-chains with DP 10–50, and only traces of A-chains. The P-chains of asSBE did apparently not possess any A-chains and only small amounts of the short B1-chains. However, as stated above, A-chains carrying phosphate groups cannot be identified since they are longer than 2–3 residues and this could be the case for the P-chains. Hence, suppression of GWD increased amylose but had no influence on the general chain length and cluster structure. Likewise, the Nchains in asSBE were of the same lengths as the ones in WT and asGWD indicating that the structure of the non-phosphorylated

Carbohydrate (relative concentration)

100

60

40

20 10

(a)

12

100

2 DP

relative

60

40

20 10

100

2 DP

(b)

60

40

20 10

2 DP

(c)

6 3

0

0.3

0.6

K av

0.9

0

of

P-

inner part of the amylopectin chains was similar in all samples. asSBE contained far more phosphate than the other samples and the P-chains were much longer. Both the external and the internal chains of asSBE were considerably longer than in the other samples. As seen in Fig. 10 there are two possibilities to obtain longer ICL values. Fig. 10a shows a schematic model where the structure contains long internal chains and all phosphate groups are located at internal segments, whereas in Fig. 10b the internal chains are shorter but a phosphate group is found at an external segment. As the ␤-amylase cannot pass this hinder the internal chain becomes apparently longer. Experimentally, it is not possible to distinguish between the two alternatives. Collected data suggest that the external chains form the double helices in crystalline lamellae, which are 5–7 nm thick [28,48]. The lamellae form stacks of alternating amorphous and crystalline layers with a repeat distance of 9–10 nm [49]. Kozlov et al. estimated the thickness of the crystalline lamellae, as well as the repeat distance for WT, asGWD, and asSBE by combining differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS) [28,50].

9

0

number

0.3

0.6

K av

0.9

0

0.3

0.6

K av

Fig. 9. Superdex 75 GPC of N-chains () and P-chains () from P-clusters of (a) WT, (b) asGWD, and (c) asSBE.

0.9

(b)

+ β-amylase

(a)

647

+ Debranching en nzymes

J. Wikman et al. / International Journal of Biological Macromolecules 48 (2011) 639–649

Fig. 10. Schematic representations of plausible structures with long P-chains in ␤-LDs; structure (a) possesses long internal chain length (ICL) and all phosphate groups (䊉) are located at the internal segments; structure (b) possesses shorter ICL but has a phosphate group at the external segment of the long B-chain. In the first step, ␤-amylase hydrolyzes susceptible external ␣-(1→4)-glycosidic bonds; in the second step the debranching enzymes hydrolyzes the ␣-(1→6)-glycosidic bonds. The products from (a) and (b) are similar in subsequent analysis by GPC.

The crystalline lamellae are 5.7 nm in WT, 5.1 nm in asGWD, and 6.7 nm in asSBE. WT and asGWD have both a repeat distance of 8.8 nm, while the distance for asSBE is much longer, 15.7 nm. This corresponds well to our ECL and ICL values. The amounts of N- vs. P-clusters correlated with the levels of phosphate in the native starches. Due to the low phosphate content in the asGWD starch, the number of P-clusters in this starch was only about one fourth as compared to the WT starch. The asGWD and the WT clusters were of similar size showing that the downregulation of the phosphorylation had not affected the structure (Table 4, Fig. 6). However, the overall cluster size of the asSBE P-clusters was considerably smaller. When the starch branching enzyme is downregulated the branching of the sample is reduced and instead longer internal B-chains are formed. As a result of the decreased branching, the branches become more separated and the groups of clustered chains smaller. The small clusters in this starch were therefore also less branched and their ICL was longer (Table 4). It is known that building blocks of groups 2 and 3 contain single and double branched dextrins, respectively. The larger dextrins of groups 4 and 5 have more than two branch points and the structures are more complicated. Group 1 contains mainly linear dextrins derived from the inter-block segments removed by the enzyme [24]. The N-clusters of WT and asGWD had similar molar distributions of groups of branched blocks where ∼15% possessed more than two branch points (Table 8). asSBE contained only ∼5% of the larger blocks, whereas group 2 predominated in this starch. This showed that the blocks in the loosely branched clusters of asSBE were mainly single branched. In addition, asSBE gave rise to the largest amounts of linear dextrins (Table 6). Using the inter-block DP (Table 6), it is possible to get a good estimation of the average inter-block CL (IB-CL, i.e., the chain length between the branches of two adjacent building blocks), which equals IB-DP + 4 [24] and was ∼6 in WT and asGWD and longer, ∼7, in asSBE (Table 8). As comparison the IB-CL in amylose-free potato is 6.9–8.9 [24]. The shorter IB-CL in WT and asGWD suggested more densely packed blocks in the clusters compared to asSBE. In the P-clusters the ratio of P6 vs. P3 was different from the native starches. This suggested that some phosphate was lost during the ␣-amylolysis, despite the fact that the phosphate groups were enriched in the cluster fractions. P-clusters in asGWD were more phosphorylated than in WT, whereas the concentration of P6 was the same in both samples, WT possessed less P3. Consequently, WT P-clusters possessed a higher ratio of P6 vs. P3, indicating that mostly P3 was lost during ␣-amylolysis. In contrast, P6 was preferentially lost from asGWD as suggested from the very low ratio. The highly phosphorylated asSBE had more phosphate in the P-clusters than WT and asGWD. As for WT, the ratio of P6 vs. P3 was high. The yield of N-chains and P-chains from the P-clusters in WT and asGWD was almost the same and the unit chain distributions were also similar, so that apparently all the A-chains were found

among the N-chains and the longer B-chains preferentially in the P-chain fraction (Table 7). asSBE, possessing the highest level of phosphate, gave the highest yield of P-chains. The lengths of the Nand P-chains were similar to those in WT and asGWD, and all Achains were associated with the N-chain population and the longer B-chains were P-chains (Table 7, Fig. 9). It was noted that, after the attack of debranching enzymes, the P-chains of the asSBE Pclusters were similar to the P-chains in the other samples. This was in contrast to the original amylopectins that possessed very different distributions of P-chains in their ␤-limit dextrins. From our data, possible principle structures of the amylopectin molecules could be drawn (Fig. 11). Though the proposed structures are based on the two-dimensional backbone model [51], which was found to explain data from the cluster analy-

P-Cluster

N-Cluster

P-Cluster

N-Cluster N-Cluster

(a) C A IB-S

N-Cluster

IC-S

IB-S

IC-S

N-Cluster

IB-S IC-S

P-Cluster

IB-S

IC-S

N-Cluster N-Cluster

(b)

C A IB-S

IC-S

IB-S

IC-S

P-Cluster N-Cluster P-Cluster

IB-S IC-S

P-Cluster

IB-S

IC-S

P-Cluster N-Cluster

(c) C

A IB-S IC-S IB-S IC-S IB-S IC-S

IB-S

IC-S

IB-S

IC-S

Fig. 11. Proposed schematic molecular structures of (a) WT, (b) asGWD, and (c) asSBE, showing the distribution pattern of internal phosphate groups. Long chains (black, bold lines), short chains (grey, thin lines), the reducing end (∅), the interblock segments (IB-S), and intercluster segments (IC-S) are indicated. WT and asGWD have structures with crystalline (C) and amorphous (A) lamellae of similar width. WT have more phosphate groups (䊉) in the internal parts than asGWD. asSBE have much thicker lamellae and the internal segments are much longer with more phosphate. Overall, the clusters are smaller in asSBE and its non-phosphorylated clusters have shorter internal segments than the phosphorylated clusters.

648

J. Wikman et al. / International Journal of Biological Macromolecules 48 (2011) 639–649

Table 8 Composition of building blocks in N-clusters of potato amylopectin. Samples

No. of blocksa

Density of blocksb

IB-CLc

WT asGWD asSBE

2.6 2.3 2.0

7.5 6.5 5.6

5.6 6.1 6.8

a b c d

Molar distribution of groups of blocksd (%) 2

3

4

5

61.4 66.3 79.8

22.5 20.5 14.9

8.6 6.8 3.4

7.5 6.4 1.9

Average number of blocks = Bbl (wt.%)/DPN-cluster /DPBbl . (No. of blocks)/(DPcluster ) × 100. Inter-block CL = IB-DP + 4. Group 2 (DP 5–9), 3 (DP 10–13), 4 (DP 14–19), and 5 (DP > 20).

ses of some other amylopectins [23,34,52], it does not exclude other alternatives. The results showed that the molecular structures of WT and asGWD were very similar besides the fact that asGWD had a much lower concentration of phosphate. Thus, in Fig. 11 structures of the N-clusters are the same in both samples. There are more phosphorylated clusters in WT; however, the structures of the P-clusters are similar in both samples. Overall, the two starches possess basically similar structures, although there is almost no phosphate in the inner segments of asGWD. asSBE had the highest concentration of phosphate in the native starch and the structural analysis showed that the downregulation of starch branching enzyme produced a starch with smaller clusters and longer internal B-chains. The proposed structure for asSBE is basically the same as for WT and asGWD, but the internal structure is stretched, much as a rubber band. As a consequence, it possesses longer internal segments between the branches, thus providing good substrates for the phosphorylating enzyme (GWD). Compared to the structures of WT and asGWD there are more P-clusters in asSBE, but the average size of the clusters is smaller. However, the lengths of the chains in the isolated clusters are rather similar in all samples. In asSBE there are more phosphorylated groups along the inner segments compared to the other two samples. Interestingly, however, the internal N-chains of asSBE amylopectin (Table 3) were not longer than the other samples, which suggest that the molecular structure of the non-phosphorylated parts of the macromolecule population was not much affected by the antisense suppression. Since it is known that GWD preferably phosphorylates the longer chains [53], the shorter chains (DP ∼10) in asSBE are probably unaffected by the antisense suppression and remains “normal”. The overall longer external and internal chains in asSBE results, however, in somewhat thicker crystalline lamellae and considerably thicker amorphous lamellae [50] as indicated in the structure model.

5. Conclusions Decreasing starch phosphate by antisense suppression of GWD had no influence on amylopectin chain length or internal and external chains. Antisense suppression of SBE, however, affected the amylopectin chain length; the phosphorylated chains of the amylopectin isolated from the asSBE potato line were significantly longer than both WT and asGWD starch. The high-phosphate sample asSBE had longer internal chain segments supposedly allowing for more efficient incorporation of phosphate. Surprisingly, the phosphorylated cluster chains of the asSBE amylopectin were very similar to the WT and the asGWD samples, showing that unit chain composition of phosphorylated clusters were independent of the overall amylopectin chain length and inner chain length. The longer ECL and ICL in asSBE affect both the crystalline and the amorphous lamellae making them thicker than in WT and asGWD.

Acknowledgements This work was funded by the European Community Marie Curie Fellowship, Stiftelsens för Åbo Akademi Forskningsinstitut, Waldemar von Frenckell’s Foundation and the Glycoscience Graduate School in Finland.

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