From the Forestry and Forest Products Research
Institute, Tsukuba Norin Kenkyu, Danchi-Nai, Ibaraki 305-8687, Japan,
the § Kyushu National Agricultural Experiment Station,
Nishigoshi, Kumamoto 861-1192, Japan, the ¶ Institut National de
la Recherche Agronomique, Institut des Produits de la Vigne, Unite de
Recherches Biopolymeres et Aromes, 2 Place Viala, F-34060
Montpellier cedex, France, and the
Complex Carbohydrate Research
Center and Department of Biochemistry and Molecular Biology, the
University of Georgia, Athens, Georgia 30602-4712
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ABSTRACT |
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The location of the 1:2 borate-diol ester
cross-link in the dimer of the plant cell wall polysaccharide
rhamnogalacturonan II (RG-II) has been determined. The ester
cross-links the apiofuranosyl residue of the
2-O-methyl-D-xylose-containing side chains in
each of the subunits of the dimer. The apiofuranosyl residue in each of
the two aceric acid-containing side chains is not esterified. The site
of borate esterification is identical in naturally occurring and in
in vitro synthesized dimer. Pb2+,
La3+, and Ca2+ increase dimer formation
in vitro in a concentration- and pH-dependent manner. Pb2+ is the most effective cation. The dimer
accounts for 55% of the RG-II when the monomer (0.5 mM) is
treated for 5 min at pH 3.5 with boric acid (1 mM) and
Pb2+ (0.5 mM); at pH 5 the rate of conversion
is somewhat slower. Hg2+ does not increase the rate of
dimer formation. A cation's charge density and its ability to form a
coordination complex with RG-II, in addition to steric factors, may
regulate the rate and stability of dimer formation in
vitro. Our data provide evidence that the structure of RG-II
itself determines which apiofuranosyl residues are esterified with
borate and that in the presence of boric acid and certain cations, two
RG-II monomers self-assemble to form a dimer.
Rhamnogalacturonan II
(RG-II)1 is a low molecular
weight, structurally complex pectic polysaccharide released from the
primary walls of plants by treatment with
endo- A single 1:2 borate-diol ester is believed to cross-link two RG-II
molecules. 1 mol of the dimer contains 1 mol of boron (3, 4, 12-14).
We have proposed that two of the four 3'-linked apiofuranosyl (Apif) residues present in dRG-II-B are cross-linked by
borate because the dimer contains approximately equimolar amounts of 3'- and 2,3,3'-linked Apif residues, whereas monomeric RG-II
(mRG-II) contains only 3'-linked Apif residues (4, 12).
However, two different Apif-containing side chains are
attached to the backbone of each RG-II monomer (chains A and B in Fig.
1). It is not known whether the borate
cross-link involves the Apif residue in one of each of the
two types of Apif-containing side chains or if the borate is
attached to the same type of Apif-containing side chains in
each monomer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1,4-polygalacturonase (1). The results of recent
studies have established that RG-II exists predominantly in the cell
walls of all higher plants as a dimer (dRG-II-B) that is cross-linked
by a 1:2 borate-diol ester (2-5). Borate ester cross-linking of pectin
may be required for the normal growth and development of plants (4-6)
because boron deficiency results in the inhibition of plant growth and
in the formation of cell walls with markedly altered physical
properties (7-11).
View larger version (14K):
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Fig. 1.
Partial structure of RG-II. The two
Apif-containing side chains (A and B) attached to the
backbone of RG-II are shown. The two disaccharides,
-l-Rhap-(1
5)-Kdo and
-l-Araf-(1
5)-Dha, which are known to be attached to
position 3 of two of the 4-linked GalpA residues, have been
omitted. The abbreviations used are Acef,
3-C-carboxy-5-deoxy-l-xylofuranosyl; Araf,
arabinofuranosyl; Arap, arabinopyranosyl; Fucp,
fucopyranosyl; Galp, galactopyranosyl; GalpA,
galactopyranosyluronic acid; GlcpA, glucopyranosyluronic
acid; 2-O-MeFucp,
2-O-methylfucopyranosyl; 2-O-MeXylp,
2-O-methylxylopyranosyl.
The ability to form dRG-II-B from mRG-II and boric acid in
vitro provides a convenient model system to examine the mechanism of dimer formation (4). In addition, such studies are likely to provide
information on the ability of dRG-II-B, which is present in fermented
beverages such as wine, to form complexes with heavy metals (15). We
have suggested that steric factors regulate dimer formation because
only divalent cations with an ionic radius >1.1 Å (e.g.
Pb2+, Sr2+, and Ba2+) increase the
rate of dRG-II-B formation significantly (4). However, the function of
cations in dimer formation has not been established, nor is it known if
the 1:2 borate-diol ester is located on the same glycosyl residue(s) in
naturally occurring and in vitro synthesized dRG-II-B. We
now report that the same two Apif residues are the sites of
borate esterification in naturally occurring and in vitro
synthesized dRG-II-B. The effects of selected mono-, di-, tri-, and
tetravalent cations on dRG-II-B formation are described as are the
abilities of Pb2+, Ca2+, and La3+
to increase the rate of dimer formation from mRG-II and boric acid.
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EXPERIMENTAL PROCEDURES |
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Isolation and Purification of RG-II-- dRG-II-B was isolated from the cell walls of sugar beet tubers (3), potato tubers (16), bamboo shoots (17), and red wine (12) as described previously.
Formation of mRG-II and dRG-II-B-- mRG-II was generated by treating dRG-II-B (50 mg) for 30 min at room temperature with 0.1 M HCl (10 ml). The solution was dialyzed (1,000 molecular weight cutoff) at 4 °C against deionized water, and the RG-II was then converted to its sodium form by elution through a column (1 × 5 cm) containing Chelex-100 (Na+ form, Bio-Rad). The eluant was then freeze dried.
dRG-II-B was generated in vitro by treating mRG-II (10 mg) for 4 days at room temperature with 50 mM potassium phthalate (10 ml), pH 3.5, containing 15 mM boric acid. dRG-II-B/Pb2+ was generated by treating mRG-II (10 mg) for 24 h at room temperature with 50 mM potassium phthalate (5 ml), pH 3.5, containing 1 mM boric acid and 0.5 mM Pb(OAc)2. The solutions were dialyzed separately (1,000 molecular weight cutoff) against deionized water and freeze dried. The boron content of mRG-II, dRG-II-B, and dRG-II-B/Pb was determined by inductively coupled plasma atomic emission spectroscopy (4) and by inductively coupled plasma mass spectrometry (3).
Methylation and Carboxyl Reduction of Methylated RG-II-- Separate solutions of mRG-II and dRG-II-B (~5 mg) in water (5 µl) were mixed with dimethyl sulfoxide (1 ml) and the mixtures purged with argon for 2 h at room temperature to remove air (18). Potassium methylsulfinylmethanide (200 µl, 2.5 M in dimethyl sulfoxide) was then added, and the solutions were kept at room temperature for 2 h. The mixtures were frozen in ice, and then methyl iodide (220 µl) was added dropwise. The solutions were kept for 2 h at room temperature to generate the methylated polysaccharide (prolonged exposure (>3 h) of the dimer to the methylating reagents resulted in partial cleavage of the borate ester). The solutions were then diluted with an equal volume of water and flushed with argon to remove the excess methyl iodide. The solutions were then dialyzed (1,000 molecular weight cutoff) for 24 h against deionized water and freeze dried. The methyl-esterified carboxyl groups of the methylated polysaccharide were carboxyl-reduced with Superdeuteride (1 ml, Aldrich) (19). The methylated, carboxyl-reduced polysaccharide was desalted by dialysis and then freeze dried.
Generation of the Rha-apiitol Derivatives by Partial Fragmentation of Methylated and Carboxyl-reduced RG-II-- The methylated, carboxyl-reduced polysaccharide (~ 1 mg) was partially fragmented by treatment for 2 h at 70 °C with 88% formic acid (300 µl). This treatment hydrolyzes the 1:2 borate-diol ester thereby exposing the hydroxyls to which it was attached and also generates a mixture of partially methylated oligoglycoses. The formic acid was removed under a flow of nitrogen gas by coevaporation with isopropyl alcohol (2 × 500 µl). The partially methylated oligoglycoses that were generated were then converted to their corresponding partially methylated oligoglycosyl alditols by treatment for 4 h at room temperature with aq. 20% methanol (350 µl) containing NaBD4 (10 mg/ml). Excess NaBD4 was destroyed by dropwise addition of glacial acetic acid, and the resulting solution was concentrated to dryness under a flow of nitrogen gas. The residue was treated with methanol containing 10% acetic acid (4 × 500 µl) and then with methanol (4 × 500 µl). The free hydroxyl groups of the methylated oligoglycosyl alditols were acetylated by treatment for 3 h at 120 °C with acetic anhydride (100 µl). Water (500 µl) was added, and the acetic acid that formed was neutralized by the addition of solid sodium carbonate. Dichloromethane (1 ml) was added, and the organic and aqueous phases were separated by centrifugation. The organic phase was concentrated to dryness, and the residue was dissolved in acetone (20 µl) before analysis by gas-liquid chromatography with mass spectrometry (GLC-MS).
Generation of the Rha-Kdo'ol Derivative by Partial Fragmentation of Methylated and Carboxyl-reduced RG-II-- The methylated, carboxyl-reduced polysaccharide (~ 1 mg) was partially fragmented by treatment for 30 min at 60 °C with 0.1 M trifluoroacetic acid (300 µl). The partially O-methylated and partially O-acetylated derivative of Rha-Kdo'ol was then generated as described previously (13, 18). The Kdo'ol generated by this procedure is a 5-substituted 1,1',2-tri-deuterio1,2,6-tri-O-acetyl-4,7,8-tri-O-methyl-3-deoxyoctitol.
Formation of dRG-II-B in the Presence of Di-, Tri-, and Tetravalent Cations-- Solutions of mRG-II (0.5 mM) in 50 mM potassium phthalate (200 µl), pH 3.5, containing 0.5 mM boric acid and a 0.5 mM concentration of one of the following salts: NaCl, KCl, MgCl2, NiCl2, Cu(OAc)2, Zn(NO2)2, SnCl2, CdCl2, CaCl2, HgCl2, SrCl2, Pb(OAc)2, BaCl2, AlCl3, SbCl3, EuCl3, PrCl3, CeCl3, LaCl3, or Ce(SO4)2.2(NH4)2SO4 were kept for 24 h at room temperature. The percentage of mRG-II and dRG-II-B present was then determined by size-exclusion chromatography (SEC) using a Superdex®-75 HR10/30 (Amersham Pharmacia Biotech Inc.) column eluted with 50 mM ammonium formate, pH 5, at a flow rate of 0.6 ml/min. The column eluant was monitored with a Hewlett-Packard 1037A refractive index detector (4).
The rate of dRG-II-B formation in the presence of Pb2+, Ca2+, and La3+ was determined by treating solutions of mRG-II (0.5 mM) in 50 mM potassium phthalate/HCl (200 µl), pH 3.7, with boric acid (1 mM) and Pb(NO3)2 (0.1 and 0.5 mM), LaCl3 (0.1 and 0.5 mM), or CaCl2 (0.5, 5, and 50 mM). The amount of dRG-II-B (calculated as µmol) that formed after 6 min, 30 min, 1 h, 2 h, and 6 h was determined by SEC (4). In a second experiment the effect of Pb2+, Ca2+, and La3+ on the rate of dRG-II-B formation at pH 5 was determined by treating solutions of mRG-II (0.5 mM) in 50 mM potassium phthalate/NaOH (200 µl), pH 5, with boric acid (1 mM) and Pb(NO3)2 (0.5 and 1 mM), LaCl3 (0.5 and 1 mM), or CaCl2 (0.5, 10, and 50 mM). The µmol of dRG-II-B which formed after 6 min, 30 min, 1 h, 2 h, and 6 h was determined by SEC.
Analytical Methods-- GLC-MS was performed with a JEOL JMS-DX303HF mass spectrometer interfaced with a Hewlett-Packard 5890 gas chromatograph. GLC-chemical ionization MS (GLC-CI-MS) was performed with ammonia as the reagent gas and a source temperature of 150 °C. GLC-electron impact ionization MS (GLC-EI-MS) was performed with an ionization current of 70 eV and an ion source temperature of 180 °C. The partially methylated and O-acetylated oligoglycosyl alditols were separated using a DB-1 column (15 m × 0.25 mm) as described (13).
11B nuclear magnetic resonance spectroscopy
(11B NMR) was performed with a JEOL JNM-A600 spectrometer
operating at 192 MHz and 25 °C (3). Samples were analyzed in quartz
NMR tubes without spinning or a field lock. Chemical shifts () are
reported in ppm relative to external boric acid (
0 ppm).
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RESULTS |
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A Chemical Procedure to Locate the Borate-esterified Apiosyl
Residues in dRG-II-B--
Two of the four Apif residues in
dRG-II-B have been shown, by glycosyl linkage composition analysis, to
be the probable sites of borate esterification (4, 12, 13). There are
three ways the borate ester can cross-link two RG-II monomers. The
borate could cross-link an Apif residue of side chain A to B
or to another A, or two B side chains may be cross-linked. We describe
in the following paragraphs experiments designed to determine which
apiosyl residues are cross-linked by the borate ester using a chemical method (see Fig. 2) originally developed
to sequence complex carbohydrates (19).
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The 11B NMR spectrum of methylated dRG-II-B in dimethyl
sulfoxide contains a signal at 8.8 which corresponds to a 1:2
borate-diol ester (2-4) and establishes that the cross-link had not
been hydrolyzed during the methylation reaction. Borate-esterified Apif residues are not methylated, whereas unesterified
Apif residues are methylated at O-2 and
O-3 (Fig. 2, step i). The methylated, carboxyl-reduced RG-II
dimer was then partially fragmented by treatment with formic acid (Fig.
2, step ii). This treatment hydrolyzes the 1:2 borate-diol ester
thereby exposing the hydroxyls to which it was attached and also
generates a mixture of partially methylated oligoglycoses including the
partially methylated disaccharide Rhap-(1
3')-Api. The
Rhap residue linked to the Apif residue from side
chain A has no O-methyl groups (Fig. 2, step i) whereas the Rhap residue linked to Apif residue from side
chain B has O-methyl groups at O-2 and
O-4 (Fig. 2, step i). This provides a way to identify the
side chain from which each Rhap-(1
3')-Api originated. The
methylated oligosaccharides were converted, by reduction with NaBD4, to their corresponding partially methylated
oligoglycosyl alditols (Fig. 2, step iii) and the free hydroxyl groups
then O-acetylated (Fig. 2, step iv).
The partially O-methylated alditol from a borate esterified
3i-linked Apif residue is acetylated at O-1,
O-2, O-3, and O-4 (see 1 in
Fig. 2), whereas the partially O-methylated alditol from an
unesterified 3'-linked Apif residue would be acetylated at
O-1 and O-4 and O-methylated at
O-2 and O-3 (see 2 and 3 in
Fig. 2). The Rhap residue originating from side chain A
would be acetylated at O-2, O-3, and
O-4 (see 1 and 2 in Fig. 2). The
Rhap residue originating from side chain B is methylated at
O-2 and O-4 and acetylated at O-3 (see
3 in Fig. 2). These Rhap-(13')-apiitol
derivatives differ in the number and positions of O-methyl
and O-acetyl groups and are distinguished by using GLC-CI-MS
to monitor their [M + NH4]+ ions
(m/z 611 (1), 555 (2), and
499 (3)). The locations of the O-methyl and
O-acetyl groups are defined by the primary fragment ions in
the GC-EI mass spectrum of the partially O-methylated and
partially O-acetylated derivative.
The Apif Residue of 2-O-MeXyl-containing Side Chain A is the Site
of Borate Esterification--
Naturally occurring dRG-II-B and mRG-II
formed in vitro were methylated and carboxyl reduced. The
Rhap-(13')-apiitol derivatives were then generated and
characterized by GLC-MS.
Two Rhap-(13')-apiitol derivatives were generated from
methylated, carboxyl-reduced dRG-II-B that were shown, by GLC-CI-MS, to
have [M + NH4]+ ions at
m/z 611 (1) and 499 (3),
respectively. The Rhap-(1
3')-apiitol derivative
1 has a molecular mass of 593 (611-18) and thus is fully
O-acetylated. The fragment ions at m/z
273 and 304 in the EI mass spectrum of 1 (Fig.
3A) confirm that the apiitol
and the Rhap residue do not contain O-methyl groups. Per-O-acetylated apiitol is the expected derivative
of a borate-esterified Apif residue. The Rhap
residue must have been substituted at O-2, O-3,
and O-4 because it contains no O-methyl groups
and thus is the expected product of the
Rhap-(1
3')-Apif of side chain A (see Fig. 1).
Taken together these data establish that the partially
O-methylated and partially O-acetylated
derivative 1 originates from side chain A in which the
Apif residue is substituted at positions 2, 3, and 3', and
the Rhap residue is substituted at positions 2, 3, and 4. Thus, in naturally occurring dRG-II-B the Apif residue of
the 2-O-MeXyl-containing side chain is esterified with
borate.
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Rhap-(13')-apiitol derivative (3) has a
molecular mass of 481 (499
18), which is the expected mass of
this monoglycosyl alditol with four O-methyl groups and
three O-acetyl groups. The EI mass spectrum of derivative
3 contains fragment ions at m/z 217 and 248 (Fig. 3C), showing that two O-methyl
groups are located on apiitol and two O-methyl groups are
located on the Rhap residue. The fragment ions at
m/z 118 and 363 establish that the apiitol is
methylated at O-2 and O-3 (see Fig.
3C) and thus originated from an Apif residue that
was not esterified with borate. The absence of the ion at
m/z 308 (aldJ1) indicates that the
Rhap residue is not methylated at O-3 and thus
must have been originally substituted at O-3 (19). No
evidence was obtained by GLC-EI-MS for the presence of the
Rhap-(1
3i)-apiitol derivative that would originate from a
borate-esterified Apif residue of side chain B. The apiitol
of such a derivative would be acetylated at O-2 and
O-3 and thus differs from derivative 3, which is
methylated at O-2 and O-3 (see Fig. 2). These
results provide strong evidence that in naturally occurring dRG-II-B
the Apif residue of the aceric acid-containing side chain
(side chain B in Fig. 1) is not esterified with borate.
Two Rhap-(13')-apiitol derivatives were generated from
methylated, carboxyl-reduced mRG-II which were shown by GLC-CI-MS to
have [M + NH4]+ ions at
m/z 499 (3) and 555 (2).
The derivative (3) with an [M + NH4]+ ion at m/z 499 corresponds to Rhap-(1
3')-apiitol and originates from an
aceric acid-containing side chain (see B in Fig. 1) that is not
esterified with borate. The derivative with a [M + NH4]+ ion at m/z 555 corresponds to Rhap-(1
3')-apiitol derivative 2 that contains two O-methyl groups. The O-methyl
groups are located on O-2 and O-3 of apiitol
since the EI mass spectrum of the derivative contains fragment ions at
m/z 419, 273, 248, and 118 (Fig. 3B).
This apiitol originates from an Apif residue that is not
esterified with borate. The Rhap residue contains no methyl
groups and must have originally been replaced at O-2, O-3, and O-4 and thus originates from the
2-O-MeXyl-containing side chain (see side chain A in Fig.
1). The results establish that neither of the two Apif
residues in mRG-II is esterified with borate. Indeed, mRG-II contains
no borate. It is important that these results demonstrate that our
chemical procedure does distinguish between borate-esterified and
unesterified apiosyl residues.
The disaccharide
-L-Rhap-(1
5)-KdopA is attached
to C-3 of one of the RG-II backbone GalpA residues (20). The
Kdo residue has cis hydroxyl groups at C-7 and C-8 which are
a potential site for borate esterification. The following experiments
were performed to determine if the Kdo is borate-esterified. The
partially methylated and O-acetylated monoglycosyl alditol
derivative of Rhap-(1
5)-Kdo was generated by partial acid
hydrolysis of methylated, carboxyl-reduced mRG-II and from naturally
occurring dRG-II-B. The GLC-CI mass spectrum of the
Rhap-(1
5)-Kdo'ol derivative contained a [M + NH4]+ ion at m/z 603 (data not shown) irrespective of whether it was generated from mRG-II
or dRG-II-B. The mass of this ion establishes that
Rhap-(1
5)-Kdo'ol did not originate from
borate-esterified Rhap-(1
5)-Kdo. No evidence was obtained
by GLC-CI-MS for a Rhap-(1
5)-Kdo'ol derivative with a
[M + NH4]+ ion at m/z
659, the mass of the ion expected for a monoglycosyl 3-deoxyoctitol
originating from borate-esterified Rhap-(1
5)-Kdo. Thus,
we conclude that the Kdo residue in RG-II is unlikely to be
cross-linked by a borate ester.
The Location of the Borate Ester Is Identical in Naturally Occurring and in Vitro Synthesized dRG-II-B-- The mechanism of dRG-II-B formation in plants is not known (4, 5). Thus the location of the 1:2 borate-diol ester may differ in naturally occurring and in in vitro synthesized dRG-II-B. We performed an experiment to determine whether the location of the borate ester is the same in naturally occurring and in vitro synthesized dRG-II-Bs.
Dimeric RG-II-B isolated from sugar beet, potato, bamboo shoots, and
red wine, dRG-II-B/Pb synthesized from mRG-II, boric acid and
Pb2+, and dRG-II-B synthesized from mRG-II and boric acid
were methylated and carboxyl reduced. The
Rhap-(13')-apiitol derivatives were generated (see Fig.
2) and then characterized by GLC-CI-MS and GLC-EI-MS. The
Apif residue of the 2-O-MeXyl-containing side
chains but not the Apif residue of the aceric
acid-containing side chains was esterified with borate in both the
naturally occurring and in vitro synthesized dRG-II-Bs
(Table I).
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Additional evidence that our chemical procedure is suitable for
determining the presence and location of borate-esterified Apif residues in dRG-II-B was obtained by the analysis of a
presumptive mRG-II generated from wine dRG-II-B. Rha-(13')-apiitol
derivative 1 (see Fig. 3A) was generated from the
methylated and carboxyl-reduced wine mRG-II, which suggested the
presence of a borate-esterified apiosyl residue. Indeed, the dimer was
shown, by SEC, to account for ~20% of the wine mRG-II (see Table I).
Taken together our results provide evidence that the borate ester
cross-links the Apif residues of the A side chains
irrespective of whether the dRG-II-B is obtained from a natural source,
synthesized from mRG-II in the presence of boric acid, or synthesized
from mRG-II in the presence of boric acid and a divalent cation.
Di- and Trivalent Cations Increase the Rate of Formation of dRG-II-B-- We have provided evidence that the location of the 1:2 borate-diol ester is identical in naturally occurring and in vitro synthesized dRG-II-Bs. Thus, the mechanism of dimer formation can be analyzed using in vitro synthesis of dRG-II-B. In a previous study we showed that only divalent cations with an ionic radius of >1.1 Å increased dimer formation in vitro (4). We now provide evidence that the ionic radius of the cation is only one of several factors that regulate dimer formation in vitro.
Divalent cations (Sr2+, Pb2+, and Ba2+) with an ionic radius >1.10 Å and trivalent cations (Eu3+, Pr3+, La3+, and Ce3+) with an ionic radius >0.90 Å significantly increased the amount of dimer formed in 24 h, whereas Ca2+ and Cd2+ (ionic radius of 0.99 and 0.95 Å, respectively) caused only a small increase in the amount of dimer formed (Table II). Somewhat unexpectedly, Hg2+, which has an ionic radius of 1.10 Å, has no discernible effect on the amount of dimer that formed (Table II). This may result from the fact that Hg2+ typically does not form stable coordination complexes with oxygen-donor ligands such as RG-II (21). Those cations that do increase the amount of dimer formed all have an affinity for oxygen-donor ligands (21). These results provide additional evidence that the cation-dependent increase in the rate of dimer formation in vitro most likely involves the formation of an RG-II-cation coordination complex. Thus, charge, ionic radius, and ligand-donor-atom selection are all factors that determine whether a particular cation will increase the amount of dRG-II-B formed in vitro.
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dRG-II-B Formation from mRG-II and Boric Acid Is Rapid in the Presence of Selected Di- and Trivalent Cations-- We showed previously that certain divalent cations increase the rate of dRG-II-B formation in vitro, although the rate achieved was relatively slow (4). We now show that dRG-II-B is formed within minutes from mRG-II and boric acid at pH 3.5 when in the presence of an appropriate divalent or trivalent cation.
Pb2+ and La3+ both induce a rapid,
concentration-dependent increase in the rate of dRG-II-B
formation at pH 3.5 (Fig. 4, A
and B). Within 5 min in the presence of 0.5 mM
Pb2+, ~55% of the monomer is converted to the dimer, and
>90% conversion occurs within 1 h (Fig. 4A).
La3+ is less effective than Pb2+ because only
60% of the monomer is converted to the dimer in 1 h, and the
conversion is ~80% after 6 h (Fig. 4B).
Ca2+ is considerably less effective than both
Pb2+ and La3+ (Fig. 4C). The rate of
dimer formation in the presence of Pb2+ and
La3+ is somewhat slower at pH 5 (Fig. 4, D and
E). Ca2+, even at high concentration (50 mM), is again considerably less effective than both
Pb2+ and La3+ at pH 5 (Fig. 4F).
Dimer formation is barely detectable within 6 h at pH 3.5 or 5 in
the absence of added cations under the conditions of our experiments
(Fig. 4, A and D), confirming that dimer
formation in vitro is pH- and cation-dependent
(4).
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DISCUSSION |
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The Location of the Borate Ester Cross-link in dRG-II-B--
We
have provided evidence that a single 1:2 borate-diol ester in dRG-II-B
cross-links the 3'-linked Apif residues of the
2-O-MeXyl-containing side chains of the two mRG-II subunits
but does not cross-link the 3'-linked Apif residues of the
aceric acid-containing side chains. The location of the B ester is the
same in dRG-II-B isolated from natural sources and synthesized in
vitro. However, 1:2 borate-apiose esters can exist in either of
two diastereomeric forms
(bis(-D-Apif)-(R)-2,3:2,3 and
-(S)-2,3:2,3-borate). Indeed, both diastereoisomers are
formed when methyl-
-D-apioside is reacted with
borate.2 It is not known if
naturally occurring and in vitro synthesized dRG-II-B
contain the same diastereoisomer. Nevertheless, we conclude that,
irrespective of the diastereoisomer formed, the structure of RG-II
itself determines which Apif residues are esterified with borate.
We have reported previously that the maximum rate of dRG-II-B formation
in vitro occurs between pH 3 and 4 and in the presence of
selected divalent cations (4). In contrast, the 1:2 borate-diol esters
of methyl--D-apioside form only above pH 5.2 even in the presence of cations.2 Thus, the pH- and
cation-dependent formation of Apif 1:2
borate-diol esters in dRG-II-B is determined by the structural
characteristics of RG-II. We propose that the charge density of side
chain A, which contains three uronosyl residues (see Fig. 1), may
explain, in part, the requirement for divalent cations in dimer
formation. Additional factors, including the conformations of both the
2-O-MeXyl- and aceric acid-containing side chains, are
likely to contribute to the specific location of the borate ester.
dRG-II-B Formation in Vitro and in Muro May Be Promoted by Different Divalent Cations-- The rate of cation-dependent dimer formation in vitro and the rate of dimer formation in suspension-cultured Chenopodium album cells are comparable.3 However, those cations that promote dimer formation in vitro (see Table II) are unlikely to be present at concentrations sufficiently high to promote dimer formation in muro (22, 23). In contrast, calcium is present in plant cell walls at mM concentrations (24), although most (>95%) of this calcium is bound, and the "free" calcium content of the wall is typically <5 mM. Furthermore, calcium ions have been reported to stabilize the borate ester cross-link in muro.3 Nevertheless, low concentrations of Ca2+ (0.5 mM) do not promote rapid dimer formation in vitro under the conditions of our experiments, although higher concentrations (>5 mM) are somewhat effective (see Fig. 4, C and F). We suggest that the roles of divalent cations in regulating borate ester cross-linking of RG-II in vitro and in muro are not the same because soluble and wall-bound mRG-II differ.
The soluble mRG-II used to form the dimer in vitro and
wall-bound RG-II are not identical. Soluble mRG-II has a backbone
that contains between 7 and 15 1,4-linked
-D-galacturonosyl residues (12, 25), whereas the
wall-bound RG-II backbone is believed to be covalently inserted within
a homogalacturonan chain (1). Calcium ions may interact with both
homogalacturonan and RG-II and thereby promote cross-link formation
in muro. Moreover, wall-bound mRG-II molecules may be
structurally constrained in a manner that favors borate ester
cross-link formation. In contrast, borate ester formation in
vitro is dependent on the direct interaction of di- and trivalent
cations with RG-II itself. This interaction is determined, in part, by
steric factors, because only divalent cations with ionic radii >1.10
Å and trivalent cations with ionic radii >0.95 Å promote dimer
formation in vitro (Table II). Di- and trivalent cations may
also stabilize the borate ester cross-link because treating naturally
occurring dRG-II-B with EDTA results in the slow but discernible
formation of mRG-II.3 Such results are consistent with a
previous report showing that calcium ions form coordination complexes
with and stabilize the 1:2 borate-diol esters of glucaric acid
(26).
dRG-II-B Is Formed by the Self-assembly of Two mRG-II Molecules-- dRG-II-B formation in muro must result from either a spontaneous self-assembly or an enzymically catalyzed process. Our results do not preclude that borate ester formation is enzymically catalyzed in muro. However, we have shown that in the presence of boric acid and certain cations, two RG-II monomers rapidly self-assemble to form a dimer and that the structure of RG-II itself may determine the location of the borate ester.
The ability of plant cell wall polysaccharides to self-assemble into
ordered structures has become the subject of considerable debate (27).
For example, the parallel arrangement of 1,4-linked -D-glucan chains in naturally occurring cellulose is
believed to result in large part from the organization of the
membrane-bound cellulose synthases because the spontaneous assembly of
parallel glucan chains is entropically unfavorable (27). The formation of ordered structures is a characteristic of homogalacturonan because
this polysaccharide spontaneously forms gels in the presence of
calcium. Cellulose formation and the calcium-dependent
gelation of homogalacturonan result from noncovalent interchain bonding (27). Moreover, a glycosyl residue in one glucan or galacturonan chain
may interact with any of the glycosyl residues in a second chain. In
contrast, RG-II dimer self-assembly requires the formation of a
covalent borate ester cross-link between the Apif residue of
the same side chain (Fig. 1, side chain A) in each mRG-II subunit. The
specificity and cation dependence of this cross-linking suggest that
there are precise structural requirements for dRG-II-B formation, and
this may explain why the structure of RG-II is highly conserved in
higher plants (1).
Borate Ester Cross-linking of RG-II May Alter the Mechanical Properties of the Plant Cell Wall-- We have provided evidence that a single borate ester cross-links two mRG-II molecules and that the location of the ester is the same in dRG-II-B isolated from different plants. The results of numerous studies suggest that the boron requirement and wall pectin content are correlated in many plants (28, 29) and that boron is required to maintain the mechanical properties of the primary wall (6, 8-11, 30, 31). For example, borate ester cross-linking of RG-II results in a rapid decrease in the wall pore size of suspension-cultured plant cells.3 Moreover, ester cross-link formation is required to prevent the walls from rupturing when the cells enter the stationary phase of their growth.3 Thus, the mechanical properties of the cell wall may be determined in large part by the macromolecular pectin network that most likely forms when RG-II is cross-linked by a borate ester. Nevertheless, additional roles for borate ester cross-linking of RG-II cannot be excluded.
In summary, we have shown that a single borate ester cross-links the
Apif residue of the 2-O-MeXyl-containing side
chain of each mRG-II subunit in naturally occurring and in
vitro synthesized dRG-II-B. Dimer formation in vitro is
pH-dependent and occurs within minutes in the presence of
certain di- and trivalent cations. RG-II is, to the best of our
knowledge, the first example of a plant cell wall pectic polysaccharide
that self-assembles to form structurally identical dimers.
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ACKNOWLEDGEMENTS |
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We acknowledge Yuko Takeda of the Forestry and Forest Products Research Institute for technical assistance. We thank Dr. Jocelyn Rose and Karen Howard of the CCRC for comments concerning drafts of this manuscript.
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FOOTNOTES |
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* This work was supported in part by United States Department of Energy Grants DE-FG05-93ER20097 and DE-FG02-96ER20220, by Hercules Inc., Wilmington, Delaware, and by Japanese Ministry of Agriculture, Forestry, and Fisheries Glycotechnology and Biodesign Program Grant BDP-98-II-1-1.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be sent: Complex Carbohydrate Research Center, the University of Georgia, 220 Riverbend Rd., Athens GA 30602. Tel.: 706-542-4419; Fax: 706-542-4412; E-mail: mao{at}ccrc.uga.edu.
2 T. Ishii and H. Ono, unpublished data.
3 A. Fleischer, M. A. O'Neill, and R. Ehwald, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: RG-II, rhamnogalacturonan II; Apif, apiofuranosyl; dRG-II-B, dimeric rhamnogalacturonan-II-boron; GLC-MS, gas-liquid chromatography with mass spectrometry; GLC-CI-MS, gas-liquid chromatography with chemical ionization mass spectrometry; GLC-EI-MS, gas liquid chromatography with electron impact ionization mass spectrometry; Kdo, 3-deoxy-D-manno-octulopyranosylonic acid; Kdo'ol, 3-deoxyoctitol; mRG-II, monomeric rhamnogalacturonan II; 2-O-MeXyl, 2-O-methyl xylose; Rhap, rhamnopyranosyl; SEC, size-exclusion chromatography.
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REFERENCES |
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