Structural Characterization of a (1→4)-β-D-galactan from Cell Walls of Zea mays Shoots

Abstract

The water-insoluble fraction of Zea mays L. hybrid B73×Mo17 shoot cell walls, pretreated with purified Bacillus subtilis (1→3), (1→4)-β-D-glucan 4-glucanohydrolase and purified B. subtilis endo-(1→4)-β-D-xylanase, was subsequently treated with a glucuronoxylan xylanohydrolase preparation, all of which were obtained from a commercially available B. subtilis α-amylase (Novo Ban 120). Carbo­hydrates (about 16% of the original water-insoluble fraction of Zea shoot cell-walls) derived from the enzyme treatment contained significant amounts of galactan and (1→4)-β-D-galactobiose in addition to glucuronoarabinoxylan and neutral sugar residues-containing rhamnogalacturonan fragments. Methy­lation analysis and partial acid-hydrolysis of the isolated galactan followed by analysis of the hydrolyzate showed that the galactan consisted of about 14 (1→4)-β-consecutively linked galactose moieties.

Abbreviations

β-D-Gase, (1→3), (1→4)-β-D-glucan 4-glucanohydrolase; endo-Xase, endo-(1→4)-β-D-xylanase; GXase, glucuronoxylan xylanohydrolase; Rha, rhamnose; Fuc, fucose; Ara, arabinose; Xyl, xylose; Man, mannose; Glc, glucose; Gal, galactose; UA, uronic acid; GAX, glucuronoarabinoxylan; AX, arabinoxylan; RGA, rhamnogalacturonan; DP, degree of polymerization; Mol wt, molecular weight; g.l.c., gas liquid chromatography.

TEXT

Plant cell-walls are composed of various kinds of polysaccharides. To elucidate the structure of component polysaccharides and especially to learn the nature of cross linkages imparted by covalent and other type of bonds, we have analyzed wall fragments derived after selective dissociation of the water-insoluble Zea mays L. hybrid B73 × Mo17 (Zea) shoot cell-wall components by enzymes.¹⁾ Structural details of Zea shoot cell-wall fragments (about 6.8% of the original water-insoluble fraction of Zea shoot cell-walls) generated by Bacillus subtilis (B. subtilis) (1→3), (1→4)-β-D-glucan 4-glucanohydrolase (β-D-Gase) treatment²⁾ and of those (about 3.5%) generated by endo-(1→4)-β-D-xylanase (endo-Xase) treatment³⁾ have been reported. When the β-D-Gase and endo-Xase treated Zea shoot cell-walls were subsequently treated with a glucuronoxylan xylanohydrolase (GXase)⁴⁾ preparation derived from Novo Ban 120 (B. subtilis), significant amounts of cell-wall fragments (about 20.9% of the original water-insoluble fraction of Zea shoot cell-walls) were liberated.⁵⁾ We have offered evidence for the presence of an arabinoxylan-rhamnogalacturonan (AX-RGA) complex in the fragments generated by treatment with a B. subtilis GXase preparation.⁵⁾ In addition, we studied a 4-linked Gal-oligosaccharide associated with the fragments generated by treatment with this B. subtilis GXase preparation. This paper describes the results. Unless otherwise stated, all materials and experimental procedures used for this study were as described in previous papers.^{2) 3) 5)}

The β-D-Gase and endo-Xase-pretreated WIS-fraction was treated with B. subtilis GXase preparation and the liberated carbohydrates (1,245.4 mg as Xyl equiv.) were resolved into seven fractions (I to VII) by chromatography on DEAE-Sephadex A-25.⁵⁾ Each fraction was further resolved into larger (1) and smaller (2) mol wt fractions. Yields of all fractions (I-1 and -2 to VII-1 and -2) and sugar composition of the major fractions were determined previously.⁵⁾ Fractions IV-1 (4.6% of total fractions) and V-1 (2.8%) were used for study on AX-RGA complex as described previously.⁵⁾

When a portion of fraction I-2 (38.0% of total fractions, Ara:Xyl:Gal:Glc = 37.6:47.7:14.7:trace), the most abundant among 14 fractions, was chromatographed on Bio-Gel P-2, it was resolved into three fractions, I-2-i (DP > about 15), I-2-ii (DP = 2) and I-2-iii (DP = 1) in the carbohydrate ratios of 87:9:4 (Fig. 1). Sugar composition analysis of fraction I-2-iii, the monosaccharide fraction, revealed Ara, Xyl and Gal in the molar ratio of 92.7:6.9:0.3. Fraction I-2-i was characterized to be feruloylated-AX fragments on the basis of the results of the neutral sugar composition (Table 1) and sugar linkage composition (Table 2) analyses of subfractions (a) to (j) obtained after resolution of I-2-i by chromatography on Bio-Gel P-10 (Fig. 2). Fraction I-2-ii (yield: 205 mg as Xyl equiv.) has a DP of 2 as determined by P-2 gel-filtration chromatography. Fraction I-2-ii was purified by rechromatography on the same P-2 column, followed by preparative paper chromatography. The disaccharide (yield:12.2 mg) appeared to be homogeneous (paper chromatography R_Gal 0.70). It exhibited a [α]_D + 64.6 (C = 0.61, in water). Sugar composition analysis showed that it consisted only of Gal. Sugar linkage composition analysis showed the presence of non-reducing terminal and 4-linked Gal residues in the molar ratio of 1:1. We conclude that the oligosaccharide in fraction I-2-ii is 4-O-β-D-galactopyranosyl-D-galactose (Lit. [α]_D + 67).⁶⁾

Fig. 1.

Chromatographic profile of fraction I-2 resolved using a Bio-Gel P-2 column.

Fraction I-2 (12.5 mg as Xyl equiv. in 1 mL of water) was applied to a column (1.5 × 150 cm) of Bio-Gel P-2 preequilibrated with water, followed by filtration through the column with water. Fractions of 1 mL each were collected and assayed for carbohydrate (A₄₈₀) and phenolic compounds (A₃₂₅). The arrows on the figure index the elution positions of Blue Dextran (1), maltose (2) and glucose (3). Tubes 90‒105 (fraction I-2-i), 186‒193 (-ii) and 206‒215 (-iii) were separately combined and concentrated.

Table 1.

Yields and neutral sugar composition of subfractions (a) to (j) obtained from fraction I-2-i by Bio-Gel P-10 chromatography (Fig. 2).

Table 2.

Sugar-linkage composition^a) of fractions I-2-i-(a) to -(i) obtained in Fig. 2.

^a) The partially methylated alditol acetates obtained from the acid hydrolyzates of the methylated sample were analyzed by g.l.c. on a glass capillary column (0.25 mm × 15 m) of DB-225. The column oven temperature was raised from 140 to 200°C at 2°C/min. T connotes a terminal residue.

Fig. 2.

Chromatographic profile of fraction I-2-i resolved using a Bio-Gel P-10 column.

Fraction I-2-i (90 mg as Xyl equiv. in 2 mL of water) was applied to a column (2.3 × 93 cm) of Bio-Gel P-10 preequilibrated with 0.1 M Na-phosphate buffer (pH 6.0), followed by filtration through the column with the same buffer. Fractions of 3.1 mL each were collected and assayed for carbohydrate (A₄₈₀) and phenolic compounds (A₃₂₅). The arrows on the figure index the elution positions of Blue Dextran (Vo), dextran of mol wt 1.0 × 10⁴ (1.0 × 10⁴) and glucose (G). Tubes 37‒44 (fraction I-2-i-(a)), 45‒51 (-(b)), 52‒61 (-(c)), 62‒65 (-(d)), 66‒71 (-(e)), 72‒75 (-(f)), 76‒85 (-(g)), 86‒119 (-(h)), 120‒125 (-(i)) and 133‒135 (-(j)) were separately combined and concentrated.

Fractions I-1 (10.2% of total fractions), II-1 (24.6%), III-1 (7.5%), VI-1 (2.9%) and VII-1 (2.6%) were subjected to the sugar linkage composition analysis.⁵⁾ No significant differences in the fractions were observed and all five fractions appear to represent subunits of GAX: Non-reducing terminal Ara (24‒27%), 4- and/or 2-linked Xyl (7‒11%), 3,4- and/or 2,4-linked Xyl (41‒49%) residues were prominent structural units. In addition, sugar linkage composition analysis showed that fraction I-1 contained about 5% 4-linked Gal residues as a structural component. Therefore, isolation of the 4-linked Gal-containing polysaccharide was attempted using fraction I-1.

Figure 3 shows the Bio-Gel A-1.5m elution profile resolving fraction I-1. Fraction I-1-i, -ii, -iii and -iv consisted of Ara, Xyl, Gal and Glc in the molar ratio of 46.9:49.0:4.1:trace, of 43.4:46.7:9.9:trace, of 35.4:47.3:17.3:trace to and of 24.9:31.3:43.8:trace. The results suggest that most of the 4-linked Gal containing polysaccharide is present in the lower mol wt fractions (lower than 1.0 × 10⁴ Da). Fraction I-1-iv (17.3 mg as Xyl equiv.), prepared from fraction I-1 (62 mg as Xyl equiv.), was chrormatographed on Bio-Gel P-4 (Fig. 4(A)). Sugar composition analysis revealed that galactose of fractions I-1-iv-b to -i ranged from 40‒75% of the constituent sugars (Table 3). Faction I-1-iv-c was particularly enriched in galactose. This fraction was, therefore, rechrormatographed on the same column (Fig. 4(B)). Comparative analysis of neutral sugar composition of selected portions of fraction I-1-iv-c eluting from the Bio-Gel P-4, suggests that Ara, Xyl and Glc residues comprise the major peak in the profile. These sugars are considered to be derived from AX and/or other polysaccharides. The galactan preparation (yield: 0.3 mg) ultimately obtained by rechromatography on Bio-Gel P-4, consisted of Gal, Glc, Xyl and Ara in the molar ratio of 87.3:5.7:2.7:4.3. The galactan preparation was partially hydrolyzed with 0.1 M trifluoroacetic acid at 100°C for 1 h and the hydrolyzate was subjected to paper chromategraphy by the multiple ascending method using 1-butanol-pyridine-water (6:4:3, v/v). Galactose (R_f 0.46) and oligosaccharides having R_f values 0.32, 0.21 and 0.14 were detected as the major components. The major R_f/(1-R_f) of predominate spots on the chromatogram, when plotted against the DP, yielded a linear regression suggesting that these sugars belong to a homologous series.⁷⁾ The R_f value of disaccharide corresponds to that of β-(1→4)-galactobiose obtained in earlier experiments. Furthermore sugar linkage analysis of the galactan preparation showed that it consisted mainly of a non-reducing terminal and 4-linked Gal residues in the approximate molar ratio of 1:13. Chromatography of the galactan preparation on a calibrated Bio-Gel P-4 column indicated an average mol wt of 2.3 × 10³ Da by comparison to standard sugars. We conclude that the isolated galactan possesses a linear β-(1→4) backbone with a DP of about 14.

Fig. 3.

Chromatographic profile of fraction I-1 resolved using a Bio-Gel A-1.5m column.

Fraction I-1 (6.2 mg as Xyl equiv. in 1 mL of water) was applied to a column (2.2 × 52 cm) of Bio-Gel A-1.5m preequilibrated with 0.1 M Na-phosphate buffer (pH 6.0), followed by filtration through the column with the same buffer. Fractions of 1.5 mL each were collected and assayed for carbohydrate (A₄₈₀) and phenolic compounds (A₃₂₅). The arrows in the figure index the elution positions of left to right, dextrans of mol wt 1.7 × 10⁵, 7.3 × 10⁴ and 1.0 × 10⁴. Tubes 47‒60 (fraction I-1-i), 61‒71 (-ii), 72‒84 (-iii) and 85‒95 (-iv) in the profile of I-1 were separately combined, dialyzed and concentrated.

Fig. 4.

Chromatographic profile of fraction I-1-iv resolved using a Bio-Gel P-4 column (A) and rechromatographic profile of fraction I-1-iv-c obtained in Fig. 4 (A) using the same column (B).

Fraction I-1-iv (17 mg as Xyl equiv. in 1 mL of water) was applied to a column (1.5 × 73 cm) of Bio-Gel P-4 preequilibrated with water, followed by filtration through the column with water. Fractions of 2 mL each were collected and assayed for carbohydrate (A₄₉₀). Tubes 23‒26 (fraction I-1-iv-a), 27‒31 (-b), 32‒34 (-c), 35‒39 (-d), 40‒42 (-e), 43‒45 (-f), 46‒50 (-g), 51‒55 (-h) and 56-60 (-i) were separately combined and concentrated. Fraction I-1-iv-c was rechromatographed on the same column, and tubes 29‒33 were combined to give a galactan preparation. The arrows on the figure index the elution positions of Blue Dextran (1) and glucose (2).

Table 3.

Yields and neutral sugar composition of subfractions obtained from fraction I-1-iv by Bio-Gel P-4 chromatography (Fig. 4 (A)).

β-(1→4)-galactans are generally found as side chains of RGA I, which is a major component of pectin with a backbone of alternating Rha and galacturonic acid residues and side chains that include α-(1→5)-arabinans, β-(1→4)-galactans and arabino-galactans.⁸⁾ However, the literature provides limited details concerning the structure of pectic arabinans, galactans or RGA in primary cell-walls of grasses. We offered evidence for the presence of a soluble RGA in Zea shoot cell-walls,⁹⁾ and for the possibility that GAX is covalently associated to RGA in Zea shoot cell-walls.⁵⁾ Now there is evidence for a β-(1→4)-galactan with a DP of about 14. Another sample of fraction I-1-iv was subjected to chromatography on Bio-Gel P-10 (Fig. 5). Tables 4 and 5 show the results of sugar composition and sugar linkage composition analyses of fractions I-1-iv-(a) to -(h) obtained in Fig. 5. The results show that 4-linked Gal-containing polysaccharide was present in all fractions, indicating that β-(1→4)-galactan with various DP (some exceeding 14) was present in fraction I-1-iv.

Fig. 5.

Chromatographic profile of fraction I-1-iv resolved using a Bio-Gel P-10 column.

Fraction I-1-iv (17 mg as Xyl equiv. in 1 mL of water) was applied to a column (2.3 × 93 cm) of Bio-Gel P-10 preequilibrated with 0.1 M Na-phosphate buffer (pH 6.0), followed by filtration through the column with the same buffer. Fractions of 3.1 mL each were collected and assayed for carbohydrate (A₄₈₀) and phenolic compounds (A₃₂₅). The arrows on the figure index the elution positions of Blue Dextran (1), dextran of mol wt 1.0 × 10⁴ (2) and glucose (3). Tubes 37‒44 (fraction I-1-iv-(a)), 45‒50 (-(b)), 51‒55 (-(c)), 56‒60 (-(d)), 61‒66 (-(e)), 67‒73 (-(f)), 74‒85 (-(g)) and 86‒100 (-(h)) were separately combined and concentrated.

Table 4.

Yields and neutral sugar composition of subfractions obtained from fraction I-1-iv by Bio-Gel-P-10 chromatography (Fig. 5).

Table 5.

Sugar-linkage composition^a) of fractions I-1-iv-(b) to -(g) obtained in Fig. 5.

^a) The partially methylated alditol acetates obtained from the acid hydrolyzates of the methylated sample were analyzed by g.l.c. on a glass capillary column (0.25 mm × 15 m) of DB-225. The column oven temperature was raised from 140 to 200°C at 2°C/min. T connotes a terminal residue.

Labavitch et al. previously reported the purification of β-(1→4)-galactanase from culture filtrate of B. subtilis strain WT 168 that had been grown on soybean arabinogalactan.¹⁰⁾ The purified β-(1→4)-galactanase hydrolyzed the β-(1→4)-galactan purified from citrus pectin by both exo- and endo-mechanisms. Release of the β-(1→4)-galactan and β-(1→4)-galactobiose from the Zea shoot cell-wall matrix was likely the result of action of a β-(1→4)-galactanase in a GXase preparation which was obtained from Novo Ban 120 (B. subtilis). Further studies will be necessary to clarify whether β-(1→4)-galactan is covalently associated to RGA in Zea shoot cell-walls.

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