Plant Physiol. (1998) 118: 1147-1158
Characterization of a Low-Molecular-Weight Glutenin Subunit
Gene from Bread Wheat and the Corresponding Protein That Represents a
Major Subunit of the Glutenin Polymer1
Stefania Masci*,
Renato D'Ovidio,
Domenico Lafiandra, and
Donald
D. Kasarda
Dipartimento di Agrobiologia e Agrochimica, Universita'
della Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy
(S.M., R.D., D.L.); and United States Department of Agriculture,
Agricultural Research Service, Western Regional Research Center, 800 Buchanan Street, Albany, California 94710 (S.M., D.D.K.)
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ABSTRACT |
Both high- and low-molecular-weight
glutenin subunits (LMW-GS) play the major role in determining the
viscoelastic properties of wheat (Triticum aestivum L.)
flour. To date there has been no clear correspondence between the amino
acid sequences of LMW-GS derived from DNA sequencing and those of
actual LMW-GS present in the endosperm. We have characterized a
particular LMW-GS from hexaploid bread wheat, a major component of the
glutenin polymer, which we call the 42K LMW-GS, and have isolated and
sequenced the putative corresponding gene. Extensive amino acid
sequences obtained directly for this 42K LMW-GS indicate correspondence between this protein and the putative corresponding gene. This subunit
did not show a cysteine (Cys) at position 5, in contrast to what has
frequently been reported for nucleotide-based sequences of LMW-GS. This
Cys has been replaced by one occurring in the repeated-sequence domain,
leaving the total number of Cys residues in the molecule the same as in
various other LMW-GS. On the basis of the deduced amino acid sequence
and literature-based assignment of disulfide linkages, a
computer-generated molecular model of the 42K subunit was constructed.
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INTRODUCTION |
The glutenin fraction of the gluten proteins is primarily
responsible for the viscoelastic properties of wheat (Triticum
aestivum L.) flour doughs. It consists of various types of protein
subunits that are linked together by intermolecular disulfide bonds.
These form a polymeric mixture that has a broad molecular-weight
distribution, with component polymers ranging from the dimeric forms
with molecular weights as low as 60,000, to polymers containing many
subunits with molecular weights in the millions (for review, see
Kasarda, 1989
; Wrigley, 1996). Variations in the types and amounts of
subunits correlate with quality variations among wheat cultivars,
probably by affecting the molecular-weight distribution of the glutenin polymers (Gupta et al., 1993, 1995). There are two main types of
subunits, the HMW-GS and the LMW-GS, with the former having been much
more extensively characterized than the latter.
Difficulties in characterization of LMW-GS arose because they derive
from many more genes than HMW-GS and because the subunits are somewhat
insoluble after reduction of the intermolecular disulfide bonds (which
is necessary for their purification, but which also breaks down
intramolecular disulfide bonds to expose buried hydrophobic regions).
Until recently, almost all attempts at cloning lmw-gs genes
led to DNA sequences corresponding to similar protein products that are
not representative of the major LMW-GS types; almost all had the
apparent N-terminal sequence METSCIPGL-, relatively low molecular
weights of about 35,000 or less, and a total of eight Cys residues,
including the Cys at position 5 (for review, see Shewry and Tatham,
1997; Cassidy et al., 1998).
In contrast to the apparently single type (with very minor variations)
of the LMW-GS indicated by the DNA sequencing, two main types of LMW-GS
have been defined on the basis of N-terminal amino acid sequences: the
LMW-s and LMW-m types, with the former starting with the sequence
SHIPGL-, and the latter represented by the METSHIPGL-, METSRIPGL-, or
METSCIPGL- N-terminal sequences (Kasarda et al., 1988; Tao and Kasarda,
1989; Lew et al., 1992). The LMW-s types are predominant. They also
tend to have higher molecular weights, in the approximate range of
35,000 to 45,000 relative to the LMW-m types, which seem to fall into
the wider molecular-weight range of about 30,000 to 45,000 (Lew et al., 1992). In bread wheat cultivars, the LMW-m type with the METSHIPGL- sequence was the next most abundant type of LMW-GS, followed by the
METSRIPGL- type, whereas the METSCIPGL- N-terminal sequence, typical of
the cloned sequences, appeared to be somewhat rare among the types
defined by direct protein sequencing (Lew et al., 1992).
Both LMW-s and LMW-m types are coded by genes present at the complex
Glu-3 loci (Glu-A3, Glu-B3, and
Glu-D3 in hexaploid wheat). Only partial sequence
information has been available for the LMW-s types because they seemed
almost impossible to clone. Recently, a partial DNA sequence that did
not show the Cys residue at position 5 was published (accession no.
X84960). Soon after, the complete DNA sequence of a lmw-gs
gene from durum wheat was achieved that might correspond to
either the LMW-s type or the LMW-m type without the Cys at position 5 (D'Ovidio et al., 1997). Both of these DNA-based sequences showed a
Cys codon in the repeated-domain region. An apparently homologous Cys
was found in glutenin by Köhler et al. (1993) and Keck et al.
(1995), although because it was defined by proteolytic digestion of a
residue glutenin preparation, peptide purification, and sequencing, the
exact position in any defined LMW-GS was not available.
For some years a misunderstanding about the nature of LMW-GS has
prevailed as a consequence of many investigators assuming that the
cloned LMW-m type with Cys at position 5 was typical of LMW-GS, even if
protein studies indicated otherwise. Consequently, we deemed it
important to sequence a major LMW-s-type gene and provide extensive
direct amino acid sequences for the corresponding protein to avoid
similar pitfalls. In particular, we considered it of great importance
to define all Cys residues in the primary structure of the subunit so
that those likely to form intermolecular disulfide bonds could be
determined (by comparison with disulfide-linked peptides described in
the literature). This would enable the subunit to be classified as a
potential chain extender (having two or more Cys residues that form
intermolecular disulfide bonds) or as a chain terminator (having only
one Cys available for intermolecular disulfide bond formation) during
the not-yet-understood oxidative polymerization process that gives rise
to the glutenin polymers in developing endosperm (Kasarda, 1989). A
predominance of the chain-extender types in glutenin should lead to
strong gluten with good viscoelastic properties, whereas too much of
the chain-terminator types would have the opposite effect.
In this paper we report the isolation and characterization of a
lmw-gs gene coding for a 42K LMW-s-type protein in the bread wheat cv Yecora Rojo, and show, for the first time to our knowledge, correspondence between a LMW-GS and its encoding gene through a
comparison with extensive amino acid sequences of the purified polypeptide. We also present evidence for this particular subunit being
very closely related to the 42K subunit found in durum wheat, which
plays a major role in determining quality, and discuss the structural
organization that might be responsible for this characteristic.
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MATERIALS AND METHODS |
The bread wheat (Triticum aestivum L. cv Yecora Rojo)
used in these studies was obtained from the California Wheat Commission (Woodland). Flour was milled from the wheat with a Quadrumat
Senior mill (C.W. Brabender Instruments, Inc., South Hackensack, NJ). The lot was designated CWC-141 and the quality characteristics of
CWC-141 flour have been reported previously by MacRitchie et al.
(1991).
DNA Extraction
Genomic DNA was isolated from 5 g of leaves from single
plants, as reported previously (D'Ovidio et al., 1992).
PCR Analysis
Amplifications of the gene encoding the 42K LMW-GS were performed
using primers and conditions reported by D'Ovidio (1993). Aliquots (10 µL) of the amplification products were fractionated on 1.5% agarose
gel in 1× Tris-borate-EDTA buffer following standard procedures
(Sambrook et al., 1989).
Cloning, Nucleotide Sequencing, and Computer Analysis
The amplification product of about 1.15 kb was purified from an
agarose gel using the Gene Clean Kit (Bio101, La Jolla, CA) and ligated
into the EcoRV dephosphorylated site of the pGEM-T plasmid
vector (Promega) using standard procedures (Sambrook et al., 1989).
After transformation into the Escherichia coli strain NM522,
the recombinant colonies were analyzed to verify the presence of the
1.15-kb fragment. Several recombinant clones contained an insert but
none of them was of the expected size. The insert size in the different
clones was about 50 to 200 bp shorter than the expected size. Similar
results have also been obtained using different E. coli
strains, and it was demonstrated that single deletions of 50 to 200 bp
had occurred within the repetitive domain of the different recombinant
clones (R. D'Ovidio, unpublished data). To overcome this limitation,
the nucleotide sequencing was carried out directly on the 1.15-kb PCR
product using the Thermo Sequenase radiolabeled terminator cycle
sequencing kit (Amersham). The PC/GENE computer program
(Intelligenetics, Mountain View, CA) was used to analyze the sequence
data.
Purification of LMW-GS
A fraction enriched in LMW-GS was obtained using a combination of
published procedures. HMW-GS and LMW-GS were obtained according to the
procedure of Singh et al. (1991) with the exceptions that extraction of
glutenin from the residue was performed at room temperature with 50%
(v/v) 1-propanol containing 50 mM Tris-HCl, pH 8.0, 1%
(w/v) DTT, and 4 M urea, and alkylation was omitted. HMW-GS
were precipitated according to the procedure of Melas et al. (1994) by
adding acetone up to 40% (v/v). After 10 min at room temperature and 5 min of centrifugation at 40,000g (20°C), LMW-GS were
selectively precipitated by bringing the acetone concentration up to
80% (v/v). After 10 min, centrifugation was again carried out as
described above, and LMW-GS present in the pellet were resuspended in
25% (v/v) ACN containing 0.05% (v/v) TFA and 4 M urea.
RP-HPLC was carried out with the System Gold apparatus, version 3.11, composed of the solvent delivery module 126 and UV-detector module 166 (Beckman). About 1 mg of the protein preparation was filtered through a
0.45-µm membrane and fractionated onto a semipreparative C8 column (10 mm × 25 cm; Vydac, Hesperia,
CA) equipped with an Aquapore guard column (4.6 mm × 3 cm;
Applied Biosystems). A linear gradient of 35% to 49% aqueous ACN over
25 min at a flow rate of 1.5 mL/min was used. Solvent A was water plus
0.07% TFA, and solvent B was ACN plus 0.05% TFA. The columns were
equilibrated at 50°C and proteins were detected by UV absorbance at
210 nm.
Single peaks were collected and analyzed on a mini SDS-PAGE apparatus
(Bio-Rad) according to the instruction manual. The 42K LMW-GS we have
analyzed in this paper corresponds to peak 10 described by Lew et al.
(1992).
Determination of the Number of Cys Residues in the 42K LMW-GS
The number of Cys residues was determined by comparing the
molecular weight of the alkylated 42K LMW-GS with that of the
unalkylated polypeptide by MALDI-MS. These analyses were performed by
an external service (Charles Evans and Associates, Redwood City, CA)
according to procedures described by Wu et al. (1995). Alkylation was
performed with 4-VP, as described by Lew et al. (1992). In some cases,
6 M guanidinium hydrochloride was used as an alternative to
4 M urea.
To reduce the experimental error attributable to the MALDI-MS technique
by decreasing the mass of the peptide analyzed, we used the same
procedure to analyze the peptides obtained from the 42K LMW-GS after
digestion with the proteolytic enzyme endoprotease Lys-C (Boehringer
Mannheim). Lys-C hydrolyzes specifically peptide bonds at the
carboxylic side of Lys residues. The proteolytic digestion was
performed according to the manufacturer's instructions.
Peptides obtained after Lys-C digestion were purified by RP-HPLC using
the chromatographic system described above and a water-ACN gradient
(both solvents A and B contained TFA) ranging from 32% to 46% in 35 min with a flow rate of 1.5 mL/min. The peaks collected were analyzed
by mini SDS-PAGE and N-terminal amino acid sequencing. Samples of the
resulting peptides were alkylated with 4-VP and submitted to MALDI-MS
analysis.
Detection of Cys-Containing Peptides in the 42K LMW-GS
To identify the positions of all of the Cys residues present in
the 42K LMW-GS, we initially followed the procedure reported by Egorov
(1997). About 10 nmol of the 42K LMW-GS, purified by RP-HPLC and
resuspended in a buffer containing 50 mM Tris-HCl, pH 7.6, 5 mM EDTA, and 6 M guanidinium hydrochloride,
was attached to the support thiopropyl Sepharose 6B (Pharmacia), which
specifically binds peptides with free sulfydryl groups. The support was
previously hydrated with the protein resuspension buffer. After
attachment of the protein, the support was equilibrated with 50 mM Tris-HCl, pH 8.0, and the protein was digested in situ
with the proteolytic enzyme chymotrypsin (sequencing grade, Boehringer
Mannheim), according to the manufacturer's instructions. Peptides that
did not contain Cys residues were removed from the resin by a washing
step, whereas Cys-containing peptides that remained selectively
attached to the support were subsequently detached by adding 0.5% DTT,
and collected. These latter peptides were fractionated by RP-HPLC with
a 5% to 35% ACN gradient (with TFA) for 120 min at a flow rate of 1.5 mL/min. Peaks were collected, alkylated with 4-VP, and repurified by
RP-HPLC, and the collected peaks were dried in a Speed-Vac apparatus
(Savant, Farmingdale, NY) for further characterization.
The same procedure was applied to the N-terminal portion of the 42K
LMW-GS obtained after Lys-C digestion, and this fragment was also
subjected to direct chymotrypsin digestion (without attachment to the
thiopropyl Sepharose column), under the same conditions as described
above, after alkylation with 4-VP. The chymotryptic fragments of the
N-terminal peptide were purified by RP-HPLC under the same conditions.
Amino Acid Sequencing and Composition Analysis
Protein/peptide sequencing was performed as described by Lew et
al. (1992). The 42K LMW-GS amino acid composition was compared with
that of a typical LMW-m-type subunit that had a Cys at position 5, also
purified from cv Yecora Rojo (corresponding to peak 14 of Lew et al.
[1992]) by a procedure similar to that reported above. Amino acid
analyses were performed by the Protein Structure Laboratory (University
of California, Davis) using an analyzer (model 6300, Beckman) with
methods as described by Ozols (1990).
Computer Molecular Modeling: Structure and Flexibility
A computer molecular model was based on the gene-derived amino
acid sequence, the secondary structure prediction, and the putative
intramolecular and intermolecular disulfide linkages, as defined by
Köhler et al. (1993), Keck et al. (1995), and Müller and
Wieser (1997) for homologous LMW-GS and 
-gliadins. The model was
constructed largely by methods described previously (Kasarda et al.,
1994; D'Ovidio et al., 1995; Köhler et al., 1997). Secondary structure prediction was carried out with the PHD program of Rost and
Sander (1993, 1994), version 5.94_317. A Silicon Graphics (Mountain
View, CA) Personal Iris workstation running Quanta 4.0 and CHARMm
(version 23.1) software (Molecular Simulations, San Diego, CA) was used
for the molecular modeling.
To avoid distortion of assigned secondary structure when disulfide
connections were patched in, the torsional angles of arbitrarily selected amino acids for which no secondary structure assignment was
indicated by the PHD analysis were adjusted in a serial process that
included frequent energy minimizations, to bring the Cys residues
assigned to a disulfide pair moderately close together (within about 1 nm) before turning on disulfide patching. Because of the large size of
the protein and the limitations of the semiempirical force fields in
dealing with solvent water, we did not attempt to study the effects of
hydration on our structures. Final equilibration was for 30 to 300 ps
(various models), followed by energy minimization.
Polypeptide chain flexibility for the 42K LMW-GS was predicted by the
method of Karplus and Schulz (1985), in which B-values (individual
atomic temperature factors) for the C
atoms of selected proteins of known three-dimensional structure from the protein
data bank were correlated with chain flexibility. Nearest-neighbor effects were included in the predictive method. The software used was
part of the MacVector package (Oxford Molecular Group, Campbell, CA).
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RESULTS |
PCR Amplification and Nucleotide Sequence of the Gene Encoding the
42K LMW-GS
SDS-PAGE patterns of the glutenin subunits found in the bread
wheat cv Yecora Rojo showed the presence of a 42K LMW-GS (Fig. 1A, lane 4), the mobility of which
corresponds to the main LMW-GS present in the LMW-2 type of durum wheat
(Fig. 1A, lane 1), which exhibit good quality relative to the LMW-1
types. This particular component is also present in some other
good-quality bread wheats (Fig. 1A, lanes 2 and 5) (Masci et al.,
1998). PCR analysis using primers specific for a gene encoding one of
the LMW-2 components (D'Ovidio et al., 1996) confirmed this
correspondence. The 1.15-kb fragment that has been shown to encode a
LMW-GS belonging to the LMW-2 type in durum wheat (D'Ovidio et al.,
1996) is also present in those bread wheat cultivars that show the 42K
LMW-GS, including Yecora Rojo (Fig. 1B) (Masci et al., 1998). On the
basis of these results, the 1.15-kb PCR product from the bread wheat cv
Yecora Rojo was sequenced. The nucleotide sequence revealed that the PCR product is 1144 bp long and corresponds to a lmw-gs
gene, the complete sequence of which is reported (accession no.
Y17845). The deduced amino acid sequence comprises 381 amino acids and includes the complete coding region and part of the signal peptide. The
part of the signal peptide included is similar to that reported for
other LMW-GS (Fig. 2).
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| Figure 1.
Comparison between SDS-PAGE (A) and the
PCR-amplification pattern (B) of durum wheat cv Lira biotype 45 (lane
1) and bread wheat cvs Red River (lane 2), Cheyenne (lane 3), Yecora
Rojo (lane 4), and Solar (lane 5). Both the 42K LMW-GS protein band and
the 1.15-kb PCR product (both indicated) were present in all bread
wheat cultivars except cv Cheyenne.
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| Figure 2.
Deduced amino acid sequence of the
lmw-gs gene corresponding to the 1.15-kb PCR product.
Underlines show those peptides that confirmed correspondence with the
42K LMW-GS. The peptide numbers are keyed to Figure 5. The
double-underlined sequence represents the final part of the signal
peptide typical of LMW-GS. Question marks (?) indicate unidentified
amino acids. The complete sequence is reported under accession no.
Y17845.
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The deduced protein sequence was compared with the amino acid sequence
obtained by direct sequencing of the 42K LMW-GS from the bread wheat cv
Yecora Rojo (see below). The direct sequence covered nearly 50% of the
total sequence. On the basis of this comparison, it was possible to
determine that the Ser at position 13 is the first amino acid of the
mature protein. However, it is noteworthy that the signal peptide
includes the MEN sequence (Fig. 2). The presence of an Asn residue
instead of a Thr residue, as in the MET sequence, the latter being
typical of the LMW-m type, might be the cause of the different signal
cleavages between the LMW-s and LMW-m types. Based on the
identification of the first amino acid of the mature protein, it was
possible to calculate that the 1.15-kb PCR product codes for a mature
LMW-GS, with 369 amino acid residues having a molecular weight of
42,111 and a pI of 8.31. The hydropathy profile (data not shown)
revealed the hydrophilic character of the repetitive domain and a more
hydrophobic character of the C-terminal domain and the short N-terminal
region (about 10 amino acids). The repetitive domain is composed of
repeats having the consensus sequence PPFSQQQQ. There are 25 repetitions of the consensus sequence or its variants. Although the
octapeptide repeat was the most common, hexapeptide, heptapeptide, and
nonapeptide repeats were also present, with variations in the number of
Gln residues mostly responsible for the differences (Fig.
3).
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| Figure 3.
Alignment of the amino acid repeats found in the
repetitive domain of the 42K LMW-GS encoded by the 1.15-kb PCR
product. The figure shows the regular distribution of the repeats from
amino acids 13 to 163 of the deduced mature protein. Cons, Consensus
sequence.
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To date, published nucleotide and amino acid sequence comparisons
between the 1.15-kb PCR product and the lmw-gs genes showed a high degree of homology (65%-85%) along the entire sequence, with
the main differences being located within the repetitive domain.
Similar to the other lmw-gs genes encoded at the
Glu-B3 locus (accession no. X84960; D'Ovidio et al., 1996,
1997), the 1.15-kb PCR product showed the presence of a Cys codon in the repetitive domain and seven additional Cys residues in the C-terminal domain.
Characterization of the 42K Protein and Amino Acid Sequencing of
the Cys-Containing Chymotryptic Peptides
Direct N-terminal sequencing of the 42K LMW-GS up to about 30 amino acids has been reported (Lew et al., 1992). To analyze in more
detail specific portions of the molecule and to verify the possible
correspondence with the 42K lmw-gs gene, we characterized primary structural regions of the protein that included the Cys residues.
To compare the purified 42K LMW-GS with a typical LMW-m type having Cys
at position 5 and a molecular weight of about 33,000 (peak 14 of Lew et
al., 1992), we submitted both to amino acid-compositional analysis. The
results are reported in Table I. Because
it is difficult to determine the number of Cys residues in a protein with high accuracy by compositional analysis, this approach was not
used; however, the compositional data provided useful information about
the possible structure of the two proteins. It is evident that the
major difference found between the two types of subunits was mainly in
the content of Glu/Gln and Pro, amino acids that are especially common
to the repeated sequence domain. Differences in molecular weight among
LMW-GS are likely to result from variations in the number of repeated
units in the repeated sequence domain, as also seems to be true for
HMW-GS (D'Ovidio et al., 1994).
We attempted to determine the exact number of Cys residues in the
intact 42K subunit by MALDI-MS. Because the molecular weight of the
alkylating group was 105, the results shown in Table
II indicate only seven Cys residues in
the 42K LMW-GS instead of the expected eight. However, the error
reported for MALDI-MS is 0.1%. Thus, for a protein with a molecular
weight of 42,000, there is the possibility of over- or underestimating
the number of Cys residues by about one residue. Alternatively, the
absence of one Cys residue after MALDI-MS analysis on the intact
protein might be caused by partial alkylation of the protein in the
repeated region. The molecular weight of the 42K LMW-GS, as deduced by MALDI-MS, was 42,123, which is practically identical to the value of
42,111 as determined from the deduced amino acid sequence.
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Table II.
Comparison of the molecular weights of the
4-VPalkylated 42K LMW-GS versus the unalkylated form, as
determined by MALDI-MS
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To determine with greater certainty the exact number of Cys residues,
we analyzed peptides obtained from the 42K LMW-GS after Lys-C
digestion. The decision to use this proteolytic enzyme came from the
observation that the 42K LMW-GS has only one Lys residue immediately
following the first Cys residue present in the C-terminal domain
(Fig. 2), which is characteristic of all Glu-3-coded
subunits, based on published sequences. Consequently, Lys-C digestion
should result in only two peptides, which, after fractionation, could then be analyzed independently by MALDI-MS with greater accuracy because of the smaller molecular weights. Purification of the resulting
Lys-C fragments by RP-HPLC (Fig. 4A),
SDS-PAGE (Fig. 4B), and N-terminal amino acid sequencing indicated that
the first three peaks shown in Figure 4A all exhibited the N-terminal
sequence of the molecule, whereas peak 4 exhibited a sequence
corresponding to the amino acids following the single Lys residue (data
not shown). Peak 4, then, corresponds approximately to the C-terminal domain of the protein. The presence of three N-terminal fragments after
Lys-C digestion might be attributable to minor heterogeneities of the
42K LMW-GS that were not resolved during analyses of the intact
molecule. Lew et al. (1992) reported heterogeneity at three positions
in the first 30 N-terminal amino acids of peak 10, corresponding to the
42K LMW-GS. In particular, they reported the presence of a Lys residue
at position 8, instead of an Arg residue. The SDS-PAGE analysis of the
protein corresponding to peak 2 indicated two bands (Fig. 4B, lane 2).
However, neither the MALDI-MS analyses of the fragments nor the
N-terminal amino acid sequences revealed the presence of a Lys-C
fragment lacking the first eight amino acids. Perhaps the two bands in
the SDS-PAGE pattern correspond to different conformational forms that
are not normalized in SDS solution.
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| Figure 4.
RP-HPLC separation of the Lys-C digests of the 42K
LMW-GS (A) and the SDS-PAGE pattern of the peaks collected (B).
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The major peaks 2, 3, and 4 were submitted to MALDI-MS analyses in both
the unalkylated and alkylated forms (Table
III). Peaks 2 and 3, which corresponded
in sequence to the N-terminal part of the molecule and extended
slightly into the unique sequence region to include the first Cys of
the C-terminal domain (Fig. 2), each showed mass differences for the
alkylated and unalkylated forms, corresponding to two pyridylethylated
Cys residues. Peak 4, which corresponded (according to its N-terminal
sequence) to most of the C-terminal domain, had a mass difference that
indicated six Cys residues. Accordingly, the MALDI-MS results clearly
show that eight Cys residues were present in the 42K LMW-GS.
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Table III.
Comparison of the molecular weights of the
4-VPalkylated Lys-C fragments versus the unalkylated peptides,
as determined by MALDI-MS
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To gain more information about the internal amino acid sequence of the
42K LMW-GS, this protein was analyzed by direct N-terminal amino acid
sequencing of the Cys-containing peptides obtained by covalent
attachment of the protein to the sulfydryl matrix, followed by in situ
chymotryptic digestion and RP-HPLC fractionation of the resulting
peptides. The results obtained (Fig. 5A)
defined sequences that included all of the seven Cys residues present in the C-terminal domain of the molecule, but failed to define the
expected Cys in the repeated sequence domain. Although we could offer
some speculation, we are uncertain why we were unable to recover this
particular peptide. A few positions in the sequences were found to be
heterogeneous in that two amino acids were identified during
sequencing. This is in agreement with the earlier work of Lew et al.
(1992) and Vensel et al. (1995).
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| Figure 5.
Amino acid sequences of Cys-containing
chymotryptic peptides obtained after covalent chromatography (A) and
peptides obtained after chymotryptic digestion of the Lys-C N-terminal
fragment of the 42K LMW-GS (B) (peak 2 of Fig. 4A). The
alternative amino acids at positions where heterogeneity is present are
reported.
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To find the missing Cys residue, we carried out chymotryptic
digestion of peak 2 (pyridylethylated) from the Lys-C digestion (Fig.
4A), which includes the complete repeated sequence region, and
sequenced the resulting peptides. The expected Cys residue was found
among the sequences obtained (Fig. 5B, peptides 17-20). Again, some
minor heterogeneities were found at a few positions.
All of the peptides sequenced were aligned with the amino acid sequence
deduced from the gene sequence (Fig. 2). The directly obtained peptide
sequences corresponded to about 50% of the gene-based sequence and
showed perfect agreement with it. Even where amino acid heterogeneities
were present in a peptide sequence at an occasional cycle (Fig. 5), at
least one of the amino acids found at that cycle corresponded to the
amino acid defined by the gene sequence.
Computer Molecular Modeling: Structure and Flexibility
A molecular model was constructed from the primary structure of
the 42K LMW-GS, as defined by the gene sequence. The secondary structure prediction indicated 6% or fewer of the residues in the
-helical conformation, a smaller amount of extended/strand structure, and the remainder almost entirely undefined (loop). Consequently, we applied only the predicted -helical and extended structure to the molecule. The structure was energy minimized, subject
to simulated heating to 300 K, and then equilibrated for 30 ps. By
adjusting the model (before heating and equilibration) as described in
"Materials and Methods," a final minimized, equilibrated structure
with suitable negative energy and low root-mean-square force could be
achieved that still retained, for example, nearly all of the assigned
-helical structure. Failure to carry out prior conformational
adjustments, as described, resulted in the complete loss of predicted
and assigned -helical structure surrounding the Cys residues during
disulfide patching followed by energy minimization. The resulting
speculative model is shown in a space-filling format (van der Waals's
radii) in Figure 6A for a 30-ps
equilibration. The C-terminal domain is expanded and presented in
protein cartoon format in Figure 6B to provide a better illustration of
the intramolecular disulfide bond arrangements. Although there were
small differences for the model in which equilibration had been carried
out for 300 ps, important conclusions based on the 30-ps model were not affected.
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| Figure 6.
Computer-generated molecular model of the 42K
subunit. A, Entire 42K LMW-GS shown in space-filling form (van der
Waals's radii for atoms) with all atoms shown in blue, except the
sulfur atoms of Cys or cystine side chains, which are shown in yellow.
B, Simplified model of the region containing the intramolecular
disulfide linkages and Cys-295, which presumably forms one of two
intermolecular disulfide cross-linkages. Residues 200 to 369 of the 42K
subunit are shown in protein cartoon format, which displays the
-helical structure as a helical ribbon. There is also a specific
display of selected side chains in a licorice (stick) bond format. The
sulfur atoms of the Cys and cystine residues are shown in yellow. The
main polypeptide chain is shown in red for residues 200 to 248 and in
blue from there on to the C-terminal end at residue 369. The numbers of
connected (intramolecular) Cys residues are shown.
|
|
Flexibility modeling (Karplus and Schulz, 1985) indicated a high degree
of flexibility for the entire repeating-sequence domain of the protein,
particularly for the stretches of Gln residues included in the repeats
(Fig. 7). This high degree of flexibility also applied to the repeating sequences surrounding the Cys residue at
position 43 of the mature protein sequence (beginning with the
SHIP- sequence). In addition, although at Cys-295 of the mature protein
sequence the polypeptide chain was predicted to have slightly lower-than-average chain flexibility, this short region consisting of
just a few residues was flanked by extensive regions predicted to have
considerable flexibility, largely because of a concentration of Gln
residues in these flanking regions. Gln residues in various known
structures had temperature factors that indicated a high degree of
flexibility for this residue, particularly when there was more than one
Gln in a series (Karplus and Schulz, 1985).
View larger version (20K):
[in this window]
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| Figure 7.
Flexibility of the polypeptide chain of the 42K
LMW-GS, as predicted by the method of Karplus and Schulz (1985). A
coefficient of 1.0 represents an average flexibility, and lower values
indicate less-than-average flexibility. Positive values of 1.15 seen
for some regions of the 42K LMW-GS are equivalent to highly flexible
regions of the polypeptide chain, based on the model proteins used in
the development of the predictive method. Arrows indicate the positions
of the two Cys residues in the 42K subunit that are supposed to form
intermolecular disulfide bonds.
|
|
| |
DISCUSSION |
LMW-GS are in large excess over HMW-GS in doughs, i.e. glutenin
polymers are composed mainly of LMW-GS. Because the LMW-GS are more
numerous and more difficult to purify, and because their structural
definition through nucleotide sequencing has been more problematical
than for HMW-GS, they have been poorly characterized so far. As a
consequence, no correspondence between a gene coding for a LMW-GS and a
recognized protein has been defined. Here we report the complete
nucleotide sequence of a lmw-gs gene from the bread wheat cv
Yecora Rojo. We show that peptide sequences corresponding to almost
50% of the total number (369 in the mature protein) of residues in
this particular LMW-GS were identical to the equivalent regions of the
deduced amino acid sequence from nucleotide sequencing. We also show
that the molecular weight obtained by MALDI-MS was virtually identical
to that calculated from the corresponding gene product. Furthermore,
the protein we characterized from the bread wheat cv Yecora Rojo is
strongly homologous to a protein that is clearly correlated with good
quality in LMW-2-type durum wheat cultivars.
We have defined all eight Cys residues of our 42K LMW-GS in the context
of their adjacent sequences. On the basis of homologies with Cys
residues that have been shown to form either intermolecular or
intramolecular disulfide bonds (Köhler et al., 1993; Keck et al.,
1995), we suggest that the first and seventh Cys residues in the
sequence are likely to participate in intermolecular disulfide bond
formation, whereas the remaining Cys residues are likely to form
intramolecular disulfide bonds (Fig. 6). The sequences that participate
in intramolecular disulfide linkages appear to be conserved (Shewry and
Tatham, 1997) and are readily recognized compared with the sequences
surrounding the disulfide bonds in other glutenin subunits and in
gliadins, apparently because stronger evolutionary constraints are
required for the formation of intramolecular bonds (Keck et al., 1995).
In contrast, the positions and surrounding sequences of the first Cys,
located in the N-terminal part of the molecule, and the seventh Cys,
located in the C-terminal part of the molecule, are more variable
(D'Ovidio et al., 1997; Shewry and Tatham, 1997).
It does not seem likely that conformational structure plays an
important role in the formation of intermolecular disulfide cross-linkages. There are bound to be some steric effects on sulfydryl accessibility and reactivity, but our model (see below) suggests that
the two Cys residues available for intermolecular disulfide-bond formation are in the flexible regions of the polypeptide chain, making
them about equally accessible. These latter two Cys residues would
define the 42K subunit as a linear chain extender (Lew et al., 1992)
capable of enhancing chain length during glutenin polymer formation. If
the 42K subunit is indeed a linear chain extender, this would be in
accordance with the correlation of this type of subunit with good
quality, which has been well established, at least for the equivalent
protein found in durum wheats (Carrillo et al., 1990; Masci et al.,
1995).
On the basis of these results and of those in the literature, it seems
plausible that both LMW-m and LMW-s types have two Cys residues
available for the formation of intermolecular disulfide bonds. Although
no work has been carried out on LMW-m types that do not have a Cys
residue at position 5 (e.g. those having the N-terminal sequence
METSHIPGL-), it seems likely that this type possesses a Cys residue in
the repeating sequence domain (the Cb* Cys described by Köhler et
al. [1993]) in place of the Cys at position 5. If both main types of
LMW-GS act as chain extenders, with just two Cys residues available for
intermolecular disulfide-bond formation, the positive correlation
between the relative abundance of the LMW-s types and the good end-use
quality of flours has to be attributed to their being present in
greater amounts in good-quality flours rather than to intrinsic
structural characteristics. This correlation between larger amounts of
LMW-s-type proteins and good quality is in agreement with the
difference in quality found between two biotypes of the Italian durum
wheat cv Lira. These biotypes differ at the complex locus
Gli-B1/Glu-B3 in having the poor-quality allelic variant of
LMW-GS (LMW-1) in one biotype but having the good-quality allelic form
LMW-2 in the other. Masci et al. (1995) found that these two biotypes
differ mainly in the abundance of a LMW-GS that is highly homologous to
the 42K LMW-GS described here. The hypothesis that the amount of LMW-s
types is positively correlated with good quality is also supported by the characterization of two allelic lmw-gs genes (D'Ovidio
et al., 1996), the products of which have different effects on quality. The deduced protein products from the durum lines show a difference of
only 15 amino acids within the repetitive domain, which is insufficient
to explain the different effects on quality, whereas these products
differ in quantity, with the protein product corresponding to the
"best" allele being present in a significantly greater amount.
The 42K LMW-GS has a higher molecular weight than the other
LMW-glutenin subunits, presumably because of the presence of a larger
number of repeated units. The repeating sequence in this domain
consists mainly of the core sequence PPFSQQ (D'Ovidio et al., 1997),
which appears about 25 times. This relatively consistent part of the
repeat is interrupted by a series of Gln residues (varying from zero to
three residues; see Fig. 3). These repeats are fairly regular in
character, more so than in other LMW-GS, and this characteristic might
also exert a positive influence on gluten quality, as measured by dough
strength and elasticity. In this regard, it is notable that a
correlation between the length of the repeated-sequence domain and
gluten-quality characteristics has already been reported for the HMW-GS
(Anderson et al., 1996). We suggest that these longer repeated regions
in LMW-GS enhance the contributions to dough strength and elasticity
relative to subunits with smaller-sized repeated-sequence domains.
The molecular model illustrated in Figure 6A did not lead to any
indication of regular structure for the repeated-sequence region.
Secondary-structure prediction left this region in the default
(unpredicted) category, although we should point out that the PHD
program does not attempt to define turns that are probably dependent on
tertiary interactions for stability to a considerable extent (Yang et
al., 1996). All secondary structure prediction is somewhat uncertain in
the absence of exact three-dimensional structural information from
physical methods such as x-ray diffraction or NMR analysis, both of
which seem largely inapplicable to gluten proteins, but it seems safe
to say that, in general, -helix prediction has a higher probability
of accuracy than 
-strand prediction and turn prediction. The
situation is complicated further by the absence of more than weak
similarities between gluten protein primary structures and proteins of
known three-dimensional structures in the protein data bank.
We suggest that the stretches of Gln residues in the repeats will
likely be largely unordered and flexible in solution, as was found by
Altschuler et al. (1997) for peptides containing strings of Gln
residues. The conclusion that the Gln residues in the repeating
sequences will be flexible and unordered was also supported by the
chain-flexibility prediction (Karplus and Schulz, 1985). The finding
that a 10-residue Gln insert into a model protein was unstructured and
highly dynamic (Ladurner and Fersht, 1997) is also in accord with our
suggestion. The potential for the non-Gln part of the sequence to form
turn structures is unlikely to be of any great importance, because
tight turns are not highly stable in the absence of significant hairpin
character in the adjacent strands or in the absence of favorable
tertiary structure (Yang et al., 1996). Also, the dihedral angle
limitation for Pro combined with the Pro-Pro motif of the repeats does
not favor tight turns, but, rather, a shallow turn angle corresponding to a somewhat extended conformational structure, analogous to the Pro
residues in a left-handed polyproline II-type helix (Toumadje and
Johnson, 1995).
Our molecular model showed fairly frequent inverse -turns that had
formed spontaneously during energy minimization and equilibration, but
these shallow turns did not promote regular structure in the repeated
region. The stability of these three-residue turns in a hydrated
structure is unknown.
The irregular amounts of Gln in the repeats, ranging from two to five
residues, may be functional by way of a tendency to prevent the
polypeptide chain from forming any highly regular intra- and
intermolecular interactions. If all repeats had exactly the same number
of Gln residues, there would be some possibility of the formation of a
large-diameter spiral, but the varying lengths of Gln residues that
include both odd and even numbers of residues will tend to diminish the
likelihood that such a regular structure might form. For the purposes
of storage-protein deposition in the endosperm cells and, ultimately,
enzymatic degradation of the storage proteins upon germination of the
seed, highly regular repeats involving Gln (with its strong tendency to
act as both hydrogen bond donor and acceptor) might lead to strongly
interacting aggregates having excessive insolubility and enzyme
inaccessibility. There is some indication that highly regular,
identical repeats that include a series of adjacent Gln residues tend
to be poorly insoluble in aqueous solutions (D.D. Kasarda, unpublished
results). These variations in the number of Gln residues per repeat
might simply reflect tolerated drift in the DNA-replication process, but there is also the possibility that irregular repeats serve to
stabilize the total number of repeats in the domain insofar as exact
repeats might be more prone to expansion and contraction during DNA
replication.
When high concentrations of proteins are present, as in water-flour
doughs, the Gln stretches in the somewhat extended repeated-sequence domain are likely to interact intermolecularly with their counterparts in other protein molecules through side-chain- and main-chain-amide hydrogen bonding. This type of interaction, modulated by interaction with water molecules as the plasticizer and with the monomeric gliadin
proteins, would very likely contribute positively to the viscoelasticity of the system (Belton, 1994).
In contrast to the large repeated-sequence domain, the intramolecularly
disulfide-bonded C-terminal domain occupies a relatively small, compact
part of the overall protein molecule in our model (Fig. 6B). Folding
that gives rise to the intramolecular disulfide bonds may be relatively
straightforward. Examination of the model suggests the following
scenario: folding of the shortest loop (C[220]-C[240]) gives rise
to the first disulfide bond, which places C(212) in a position to
interact with C(247), leaving three Cys residues of the C-terminal
domain unreacted. C(295), which apparently forms an intermolecular
disulfide bond, is located in a somewhat flexible loop, perhaps
stiffened by some extended structure. Flexibility in the region of
C(295) was supported by the flexibility prediction. In any case, we
postulate that there is sufficient structure to this loop to make it
impossible for C(295) to access conformational space in the vicinity of
either C(248) or C(345). Accordingly, these latter two Cys residues, which are homologous to certain Cys residues of related subunits and
gliadins, which are known to oxidize to form an intramolecular bond,
eventually find one another in conformationally allowed space and form
the third intramolecular disulfide bond. To what extent disulfide-bond
formation is catalyzed by protein disulfide isomerase, chaperones, or
other types of proteins is not well defined. All of the predicted
-helix in the molecule seems to be located in the vicinity of the
intramolecular disulfide bonds. There is at least the possibility that
helix-helix interactions are involved in guiding the formation of the
intramolecular disulfide bonds, although such interactions were not
evident in our model.
Finally, it is noteworthy that the nucleotide sequence of the
lmw-gs reported here also includes the codon corresponding
to an Asn residue instead of the codon for a Thr residue. This gives rise to an MEN sequence in our subunit, which is homologous to the MET
sequence typical of the N terminus of the LMW-m-type subunits. So far,
no LMW-m type having the MEN sequence instead of the MET sequence has
been reported (for review, see Shewry and Tatham, 1997; Cassidy et al.,
1998). Because, in the subunit we describe, the MEN sequence is part of
the signal peptide rather than corresponding to the N-terminal
sequence, as in LMW-m types, but is close to the signal cleavage site,
it may be speculated that differential processing occurs between the
LMW-m and LMW-s types because of the presence of the Asn in the MEN
sequence. This differential processing might give rise to the slightly
different N-terminal sequences characteristic of LMW-m versus LMW-s
types.
Nevertheless, the classification of LMW-GS as the LMW-m and LMW-s types
probably has no essential importance in itself with regard to dough
quality. The main difference among the various LMW-GS resides in
the presence of the first Cys residue either in the short,
nonrepetitive N-terminal region or farther along in the repetitive
domain, but all seem likely to have two Cys residues available for
intermolecular disulfide-bond formation, are chain extenders, and form
linear, as opposed to branched, polymers. The likely similarity of both
types in having two Cys residues available for intermolecular
disulfide-bond formation, one located near the N terminus and the other
located near the C terminus, probably predominates in determining how
these similar types of subunits affect properties significant to good
quality.
| |
FOOTNOTES |
1
This research was supported in part
by the Italian Ministero delle Risorse Agricole, Alimentarie Forestali,
National Research Project "Plant Biotechnology," the Italian
Ministero per l'Universita' e la Ricerca Scientifica e Tecnologica,
and the National Research Project "Studio delle proteine dei cereali
e loro relazioni con aspetti tecnologici e nutrizionali."
*
Corresponding author; e-mail masci{at}unitus.it; fax 761-357242.
Received May 18, 1998;
accepted August 24, 1998.
| |
ABBREVIATIONS |
Abbreviations:
ACN, acetonitrile.
HMW-GS, high-molecular-weight
glutenin subunit(s).
LMW-GS, low-molecular-weight glutenin subunit(s).
MALDI-MS, matrix-assisted laser-desorption ionization-MS.
RP-HPLC, reversed-phase HPLC.
TFA, trifluoroacetic acid.
4-VP, 4-vinylpyridine.
| |
ACKNOWLEDGMENTS |
We thank Donald D. Kuzmicky (Western Regional Research Center
[WRRC]) for carrying out the amino acid-sequencing analyses, Sam
Huang (California Wheat Commission) for supplying and milling the wheat
sample, Marco Spigaglia (DABAC, Viterbo, Italy) for technical
assistance, Dave Rockhold (WRRC) for carrying out the MacVector program
analysis, and Olin D. Anderson and Susan B. Altenbach (WRRC) for
helpful discussion.
| |
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Top
Abstract
Introduction
