From the Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0406
Received for publication, September 28, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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In certain maize genotypes (nulls),
In certain maize genotypes (nulls), We have identified BGAF as a protein belonging to the jasmonic
acid-induced protein (JAp) family, and BGAF is solely responsible for
In maize, cDNAs corresponding to the Cloning and Expression of Parental and Chimeric Purification of Purification of BGAF--
Etiolated seedlings of 3-day-old maize
inbred line H95 (null) was used as the source for purification of BGAF.
BGAF was isolated in its free form from the pellet fraction of shoot
homogenates that had been extracted four times with 50 mM
NaAc (pH 5.0) buffer containing 30% AS (these conditions keep BGAF
insoluble but removes Cloning and Sequencing of the BGAF cDNA--
The N-terminal
sequence of BGAF purified as described above was:
(V/X)(I/E)(G/P) (N/L)YAPIGIGATV. The peptide
APIGIGAT was used to design two (sense) degenerate primers (BGAF-6,
CCNATHGGNATHGGNGCNAC; BGAF-7, GCNCCNATHGGNATHGGNGC). Messenger RNA was
isolated from etiolated 2-day-old H95 shoots using oligo-dT-coated
magnetic beads according to the vendor's protocol (Dynal). An oligo-dT primer was used for first-strand cDNA synthesis with avian
myeloblastosis virus-reverse transcriptase (Promega). To amplify the
BGAF cDNA, the primers BGAF-6 and BGAF-7 were individually paired
with the oligo-dT primer in separate PCR reactions. A PCR product of 1 kb was obtained, gel purified, and cloned into the SmaI site
of pBluescript II SK (±) for sequencing. A BLAST search of the maize EST data base "hit" three ESTs whose 3'-ends overlapped with the 5'-end of the BGAF cDNA. Of these, the longest one (459 bp,
GenBankTM T70648) had the highest match (97%) and a 327-bp overlap
with the 5'-end of the BGAF cDNA. A primer (BGAF-16,
5'-CAGCTCCTCATCTCAAGTGTG-3') was derived from the extreme 5'-end of the
EST T70648 and used with an extreme 3'-end primer (BGAF-12,
5'-CGATTCAAGTGCCAATCTCTGGC-3') to amplify, clone, and sequence the
longest possible cDNA from H95 by RT-PCR.
Expression of BGAF cDNA in E. coli--
The 1118-bp H95 BGAF
cDNA sequence was utilized to design oligonucleotides to synthesize
cDNAs encoding the mature (BGAF-13, sense,
5'-CATATGGCTAGCGTCATAGACAACAAGGCGCCG-3'; BGAF-12,
antisense, 5'-CTATCTCGAGCGATTCAAGTGCCAATCTCTGGC-3') and the
precursor (BGAF-15, sense,
5'-CATATGGCTAGCATGGCCAGCCTCCAAGTGACTCC-3'; BGAF-14,
antisense, 5'-CTATCTCGAGTCACAGGGATCGCACGTAAACG-3') BGAF
polypeptides for expression in E. coli. The cDNAs
were cloned into the expression vector pET21a. The E. coli
host strain pLys S was transformed with the recombinant plasmid
construct and used in expression studies. Expression of BGAF from the
putative BGAF cDNA was corroborated by functional binding assays as
described previously except crude BGAF expression extracts were used to
incubate with purified Glu1. Additionally, BGAF expression extracts
were electrophoresed on 12% (w/v) SDS gels (26) and electroblotted
onto a polyvinylidene difluoride membrane for immunostaining. The
membranes were incubated overnight with rabbit anti-BGAF serum. The
rabbit anti-BGAF IgG (1° antibody) binding was detected with
peroxidase-conjugated goat-anti-rabbit IgG (2° antibody) using the
peroxidase substrate 4-chloro-1-naphthol.
BGAF Binding Assays--
To examine the molecular basis of the
BGAF-
When we tested intact Glu1 and Glu2 and a Glu2/Glu1 chimera (C-3) for
BGAF binding, the gel-shift assay yielded positive binding results as
evident from the formation of BGAF-
In contrast, a Dhr1/Glu1 chimera (C-5) and a Dhr1/Glu2 chimera (C-6) in
which the extreme N-terminal 29-amino acid-long segments of Glu1 and
Glu2 were replaced with their Dhr1 homologue bind BGAF, indicating that
this segment is not involved in BGAF binding (Fig. 2, lanes
22 and 24). However, C-5, which had the highest electrophoretic mobility (Fig. 2, lane 21), produced
predominantly two distinct bands after interaction with BGAF (Fig. 2,
lane 22). The region spanning amino acids
Glu50-Phe205 was bisected through the
construction of chimera C-26 in which the N-terminal
Ser1-Asn127 region of Glu1 was replaced with
its Dhr1 homolog. Interestingly, C-26 did not have any BGAF binding
activity (Fig. 2, lane 28) similar to C-19 (Fig. 2,
lane 26), which establishes that the polypeptide segment
spanning amino acids Glu50- Asn127 must contain
the other region(s) of Glu1 or Glu2 that is involved in forming the
BGAF binding site. Binding assays show that BGAF does not bind to Dhr1
(Fig. 3, right panel) where the results were similar to
those obtained with negative control BGAF by itself and BGAF plus
E. coli lysate (data not shown). In contrast, the amount of
BGAF detectable by immunoblotting decreased as the amount of BGAF
reacting with the positive tester Mapping BGAF Binding Regions--
Our structural analysis relative
to mapping was on the three-dimensional structure of Glu1 resolved by
x-ray crystallography in collaboration with Bernard Henrissat's
crystallography group in Marseilles, France (27). Our initial finding
that two regions (28 and 17 amino acids long) within the C-terminal 47 amino acids (based on data with C-15 and C-16) were each essential, but
not sufficient, for BGAF binding led to the analysis of these regions on the surface of the Glu1 three-dimensional structure. It appears that
the extreme 17-amino acid-long C-terminal region alone makes a greater
contribution to BGAF recognition and binding than the 30-amino
acid-long C-terminal region preceding it (cf. Fig. 2, lanes 12 and 14). To identify other polypeptide
regions that are involved in BGAF binding, we scanned structural
elements and amino acids located on the surface in the direct vicinity
of the C-terminal 17 amino acids. Analysis of the three-dimensional
structure of Glu1 indicates that the N-terminal region maps proximally
to the C-terminal region (Fig. 4). On
this basis, chimeras C-5, C-6, C-19, and C-26 were tested for BGAF
binding activity. Chimeras C-5 and C-6 had binding activity, whereas
C-19 and C-26 did not. Collectively, these binding data indicate that
the 77-amino acid-long region comprising amino acids
Glu50-Asn127 in the N-terminal half contain the
other determinant(s) that is (are) involved in BGAF binding, and they
map to the surface proximal to certain residues from the C-terminal
47-amino acid-long region. Both the binding data and the structural
data corroborate the postulate that binding requires the formation of a
site through folding by two distant regions of the primary
structure.
Isolation and Identification of the BGAF cDNA--
The BGAF
cDNA was cloned and sequenced as described above and reported
(GenBankTM accession number AF232008). The longest BGAF cDNA
isolated from H95 is 1118 bp long and includes a 918-bp coding sequence
and a 43-bp 5'- and a 157-bp 3'-untranslated region. Fig.
5 shows the deduced primary structure of
the 306-amino acid-long putative BGAF precursor, which contains two
octapeptide (G(P/R)WGGSGG) repeats that are separated by 40 amino
acids. These two repeats are postulated to play an essential role in
forming the sites involved in binding to Expression of the BGAF cDNA in E. coli--
Two BGAF cDNA
expression constructs (one coding for the putative BGAF precursor and
the other for the mature protein) were prepared and expressed in
E. coli. The mature protein encoding BGAF cDNA was
expressed in moderate amount, and it yielded a protein whose
electrophoretic mobility was faster than BGAF isolated from plant
tissue (Fig. 6A, lanes
3 and 4). Yet, this smaller (27.5 kDa) recombinant BGAF
protein was still able to bind and aggregate The The BGAF- The finding that chimera C-19 did not bind BGAF suggested that the
N-terminal 205 amino acids contain the other region(s) that is
(are) involved in BGAF binding. Analysis of the three-dimensional structure of Glu1 (27) indicated that several segments from the
N-terminal 205-amino acid-long region are located on the surface proximal to the C terminus (Fig. 4). The BGAF binding data from C-5,
C-6, C-19, and C-26 together bracketed the region spanning Glu50-Asn127 as the other region contributing
to the structure of the BGAF binding site. Again, within this region,
Glu1 and Glu2 differ from Dhr1 by 13 amino acid substitutions. Of
these, four sites (I72V, N75D, K81A, and T82A; highlighted
in purple in Figs. 1A and 4) are likely to make a
major contribution to the formation of the BGAF binding site, because
they all cluster within a surface patch (Fig. 4). The patch includes
four amino acids (N483G, T485E, Y487T, and E490R; purple in
Figs. 1A and 4) from region
Phe466-Lys493 and 2 (K496Q and T500G;
purple in Figs. 1A and 4) of six from region
Lys496-Ala512 from the extreme 47-amino
acid-long C-terminal region. Four sites (K502A, P504K, S505N, and
K506N) at which Glu1 and Glu2 differ from Dhr1 are not shown in Fig. 4,
because they are within the last 11-amino acid-long free coil region at
the extreme C terminus that could not be resolved in the crystal
structure. Thus, the binding assays have identified two discontinuous
segments (Glu50-Asn127 near the N terminus and
Phe466-Ala512 at the C terminus) that are
brought together on the surface of the The N-terminal sequence data from a purified BGAF preparation and the
three maize ESTs whose 3'-end overlapped with the 5'-end of the cloned
BGAF cDNA were key to the isolation of a cDNA (1118 bp) with a
full-length coding sequence from the maize inbred H95. The fact that
the experimentally determined N-terminal sequence (VISNKAPIGIGATV)
starts 38 amino acids after the first methionine in the deduced primary
structure of the putative BGAF precursor suggests that the cDNA
contains a full-length (918 bp) coding sequence (Fig. 5). Thus, the
BGAF precursor is 306 amino acids long and has a 38-amino acid-long
signal peptide. Thus, our experimentally determined N-terminal sequence
belongs to a 268-amino acid-long mature BGAF protein whose calculated
molecular mass and that of the one expressed in E. coli are
similar (27.5 kDa) but is smaller than expected (~35 kDa), suggesting
that the native BGAF isolated from maize is post-translationally
modified (e.g. glycosylated). Interestingly, BLAST searches
indicated that BGAF shared significant amino acid identity (58 to 61%)
with three small heat-shock proteins (GenBankTM AF021258, U43496, and
U43497) from barley that function in the systemic acquired resistance
response and with a similar protein (GenBankTM U32427 (31)) from wheat
(47% identity), which is also involved in systemic acquired resistance.
Immunoblots of E. coli lysates, in which the precursor BGAF
and mature BGAF protein coding clones were expressed, provided unequivocal identification of the isolated BGAF cDNA. The blots showed that an immunoreactive band with the same electrophoretic mobility and molecular size as BGAF isolated from plant extracts in the
case of the precursor (Fig. 6A, lanes 5 and
6) and a smaller than expected polypeptide in the case of
the recombinant mature protein (Fig. 6A, lanes 3 and 4). In addition, functional assays confirmed the
presence of BGAF in E. coli expression extracts, which
tested positive for A most intriguing feature in the primary structure of BGAF was the
presence of two octapeptide repeats (G(P/R)WGGSGG), which occur 40 amino acids apart (Fig. 5) and whose variants are also present in three
barley JAps as GPWGG(N/S)GG and in one wheat JAp as
GPWG(K/G)(I/P)(S/C)G. We postulate that each of these octapeptides either individually forms a -glucosidase does not enter the gel and therefore cannot be detected
on zymograms. Such genotypes were initially thought to be homozygous
for a null allele at the glu1 gene. We have shown that a
-glucosidase aggregating factor (BGAF) is responsible for the null
phenotype, and it specifically interacts with maize
-glucosidases
and forms large insoluble aggregates. To understand the mechanism of
the
-glucosidase-BGAF interaction, we constructed chimeric enzymes
by domain swapping between the maize
-glucosidase isozymes Glu1 and
Gu2, to which BGAF binds, and the sorghum
-glucosidase (dhurrinase)
isozyme Dhr1, to which BGAF does not bind. The results of binding
assays with 12 different chimeric enzymes showed that an N-terminal
region (Glu50-Val145) and an extreme
C-terminal region (Phe466-Ala512) together form
the BGAF binding site on the enzyme surface. In addition, we purified
BGAF, determined its N-terminal sequence, amplified the BGAF cDNA
by reverse transcriptase-polymerase chain reaction, expressed it in
Escherichia coli, and showed that it encodes a protein
whose binding and immunological properties are identical to the native
BGAF isolated from maize tissues. A data base search revealed that BGAF
is a member of the jasmonite-induced protein family. Interestingly, the
deduced BGAF sequence contained an octapeptide sequence
(G(P/R)WGGSGG) repeated twice. Each of these repeat units is
postulated to be involved in forming a site for binding to maize
-glucosidases and thus provides a plausible explanation for the
divalent function of BGAF predicted from binding assays.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Glucosidase (
-D-glucoside glucohydrolase, EC
3.2.1.21) occurs ubiquitously in all three (archaea, eubacteria, and
eukarya) domains of living organisms. The enzyme catalyzes the
hydrolysis of aryl and alkyl
-D-glucosides as well as
glucosides with a carbohydrate moiety such as
-linked
oligosaccharides (1). The occurrence and activity of
-glucosidase in
maize is correlated with growth and certain desirable traits (2). The
major function of maize
-glucosidase, however, may be in the defense
of young plant parts against pathogens and herbivores by releasing
toxic aglycones (e.g. hydroxamic acids) from their
glucosides. Hydroxamic acids, derivatives of 1,4-benzoxazin-3-one, are
believed to be the major defense compounds in maize, wheat, rye, and
wild barley (3). The predominant hydroxamic acid glucoside in maize is 2-glucopyranosyl 4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOAGlc), whose aglycone DIMBOA is the primary defense chemical against aphids
and the European corn borer (Ostrinia nubilalis). Several studies have shown a high correlation between DIMBOA content of maize
genotypes and the level of resistance to or inhibitory affect on
insects and pathogens (4-8).
-glucosidase occurs as part of
large insoluble quaternary aggregates (9). The
-glucosidase zymograms of such genotypes are devoid of enzyme bands (10). These
genotypes were initially thought to be homozygous for a null allele at
the glu1 gene. However, biochemical and immunological data
from our laboratory established that the so-called null genotypes have
-glucosidase activity when assayed in solution, and have a 60-kDa
polypeptide reacting specifically with anti-
-glucosidase sera on
immunoblots (9). The enzyme is not detected on zymograms, because it
occurs as large quaternary structures (>1.5 × 106
Da), which fail to enter the gel. After dissociation of these structures by SDS, the enzyme can be detected on gels (11). We have
recently shown that the null phenotype is due to a
-glucosidase aggregating factor (BGAF),1
which specifically interacts with the enzyme, forming high molecular weight heterocomplexes (11).
-glucosidase aggregation and insolubility, and thus, the apparent
null phenotype. Jasmonic acid and salicylic acid are plant-signaling
molecules that play an important role in induced disease resistance
pathways. JAps are believed to function in some step of these pathways.
Blocking the response to either of these signals can render plants more
susceptible to pathogens (12-17) and insects (18). Recently, it was
shown that the jasmonic acid-dependent induced systemic resistance
pathway and the salicylic acid-dependent systemic acquired resistance
pathway are fully compatible, and they result in an additive effect on
the level of induced protection (19).
-glucosidase genes
(glu1 and glu2) have been cloned and sequenced
(Refs. 20, 21; A. Esen and M. Shahid, direct submission GenBankTM
accession number U25157). The putative protein products of these
cDNAs, Glu1 and Glu2, show 90% sequence identity with each other.
Additionally, a
-glucosidase cDNA (dhr1) from sorghum
has been sequenced, which shares 70% sequence identity with Glu1 and
Glu2 (22). Despite their high sequence identity, the maize and sorghum
enzymes are functionally different with respect to BGAF binding. BGAF
binds to both maize isozymes (Glu1 and Glu2) with high specificity but does not bind to their sorghum homolog Dhr1. Therefore, they provide an
excellent system to study functional differences at nonconserved residues and elucidate the mechanism of BGAF-mediated enzyme
aggregation and insolubility. The objective of the present study is to
elucidate the mechanism of the
-glucosidase-BGAF interaction. To
this end, we have generated a series of chimeras among Glu1, Glu2, and
Dhr1 by domain swapping to identify the sites involved in BGAF binding. The binding properties of these chimeras enabled us to identify two
separate and distinct polypeptide segments that together form a BGAF
binding site on the surface of maize
-glucosidases. Finally, we have
cloned and sequenced the BGAF cDNA and confirmed its identification by expressing it in Escherichia coli and demonstrating the
activity of its recombinant protein product unequivocally in binding assays.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Glucosidase
cDNAs--
The cloning and expression of cDNAs encoding Glu1,
Glu2, and Dhr1 in E. coli were as described by Cicek and
Esen (23). The cDNA encoding the mature sorghum
-glucosidase
(dhurrinase-1) protein was amplified by polymerase chain reaction (PCR)
using the same procedure and primers (sense,
5'-GCCACGAGCTAGCACGATAAGCAGTGAG-3' and antisense,
5'-AGCAGCTCGAGTTCTACTTAATTAGTTAAGC-3') containing NheI (5'-end) and XhoI (3'-end) restriction
enzyme sites (underlined), respectively. The
construction and the expression of chimeric cDNAs were done using
the wild-type parental plasmids as templates (i.e.
glu1, glu2, and dhr1 above). The
peptide sequences from which the oligonucleotides were derived for
chimeric cDNA construction by PCR are underlined in Fig.
1A (see below). Chimeric cDNAs (proteins shown in Fig.
1B) were constructed by the
PCR-based technique of overlap extension (24). The internal
oligonucleotides used to assemble chimeric
-glucosidases were: C-2,
(sense) 5'-GGCTACTTCGCGTGGTCTCT-3' and (antisense)
5'AGAGACCACGCGAAGTAGCC-3'; C-4, sense same as C-2 sense, and C-4
antisense same as C-2 antisense; C-15, (sense) 5'-GCCAAGTGGTTGAGGAGTTCAA-3' and (antisense)
5'-TTGAACTCCTGCAACCACTTGGC-3'; C-16, (sense)
5'-GCCAGGTGGTTGAAAGAGTTCAA-3' and (antisense)
5'-TTGAACTCTTTCAACCACCTGGC-3'; C-21, (sense)
5'-GGCATYGTCTACGTCGAYCGC-3' and (antisense) 5'-GCRRTCGACGTAGACRATGCC-3' (chimera 16 was the template in PCR); C-22, (sense)
5'-GGCATYGTCTACGTCGAYCGC-3' (chimera 16 was the template in PCR) and
(antisense) 5'-GCRRTCGACGTAGACRATGCC-3'; C-19, (sense)
5'-TGCCCCRGGRCGRTGCTCACCKGG-3' and (antisense)
5'-CCMGGTGAGCAYCGYCCYGGGGCA-3'; C-18, (sense)
5'-GGCTACTTCGCGTGGTCTCT-3' and (antisense) 5'-AGAGACCACGCGAAGTAGCC-3'; C-5, (sense) 5'-TGCCGCCACTTCAGCGTACCA-3' and (antisense)
5'-TGGTACGCTGAAGTGGCGGCAC-3'; C-6, (sense) same as C-5 sense, and
antisense same as C-5 antisense; C-26, (sense)
5'-TATGTAACAATTTTCCACTGGGA-3' and (antisense)
5'-TCCCAGTGGAAAATTGTTACATA-3'. All wild-type and chimeric enzymes
were expressed without tags to eliminate the possibility of undesirable
affects on protein structure, activity, and stability. Protein
production in E. coli pLys S cultures containing each of the
recombinant plasmid constructs and protein extraction from
cultures after induction were performed as described
previously (23, 25).
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Fig. 1.
A, sequence alignment showing the N-
(1) and C-terminal (453) regions of maize (Glu1 and Glu2) and
sorghum (Dhr1) -glucosidases. The underlined
peptides were used to design oligonucleotides to create the
chimeric enzymes shown in B. The two regions
(Glu50-Asn127 and
Phe466-Ala512) that contain the residues
forming a BGAF binding site are in green background. The
residues that form the binding site are shown in red
(invariant) and purple (variant), and those that are variant
but equivalent or located outside the postulated boundary of the site
are blue. See Fig. 4 for details. B, schematic
representation of the 12 chimeric
-glucosidase constructs used to
identify BGAF binding domains in maize (Glu1 and Glu2) and sorghum
(Dhr1)
-glucosidases. The results from BGAF binding assays (+ for
binding and
for not binding) are summarized under the column
BGAF binding. The lengths of the domains in Glu1 or Glu2
replaced with their Dhr1 homolog by domain swapping are at the
right of each chimera.
-Glucosidases--
The wild-type
-glucosidases and their chimeras (C-2, C-4, C-15, C-16, C-21, and
C-22) were isolated from lysates of 800-ml cultures grown at 37 °C
and induced at 25 °C. The enzymes were purified to near homogeneity
using a combination of differential solubility fractionation by
ammonium sulfate (AS) and hydrophobic interaction chromatography as
described previously (25).
-glucosidase). The fourth pellet containing
predominantly free BGAF was extracted with 50 mM NaAc
buffer (pH 5.0). The extract was subjected to gel filtration on
Sephacryl HR-200 (90 × 1.6 cm), and the fractions containing BGAF
were identified by enzyme-linked immunosorbent assay and pooled.
Ammonium sulfate was added to the pooled fractions to a final
concentration of 0.8 M and applied to a ToyoPearl-butyl 650 M hydrophobic interaction chromatography column
equilibrated with 50 mM NaAc (pH 5.0) containing 0.8 M AS. The column was developed with a manual step gradient
using 0.1 M increments from 0.8 to 0.1 M AS.
BGAF-containing fractions were determined by enzyme-linked
immunosorbent assay, pooled, and concentrated on a 10,000 cut-off spin
column (Gelman Sciences). The purity of BGAF was checked by SDS-PAGE,
and ~250 pmol of BGAF were subjected to N-terminal sequencing.
-Glucosidase-BGAF Binding Assays--
The interaction between
BGAF and
-glucosidase was measured in a binding assay by mixing
purified BGAF at 10-fold molar excess with purified
-glucosidases or
their chimeras. In the case of chimeras C-5, C-6, C-18, C-19, and C-26,
crude expression extracts rather than purified enzymes were used as
-glucosidase source in binding assays. The BGAF-
-glucosidase
interaction is very specific and reminiscent of antigen-antibody
interactions, therefore, crude expression extracts rather than purified
enzyme could be used as ligand source. The enzyme-BGAF mixes were
incubated on ice for 1-2 h with occasional mixing. The reaction mixes
were then electrophoresed on 8% native gels, and the gels were
equilibrated with two changes of 50 mM citrate/100
mM phosphate buffer (pH 5.8) for 5 min each after
electrophoresis.
-Glucosidase activity was detected by incubating
the equilibrated gels in a 1 mM solution of the fluorogenic
substrate 4-methylumbelliferyl-
-D-glucoside (MUGlc) for
5-10 min.
-Glucosidase activity zones (bands) were visualized under
UV light and documented using an AlphaImager 2000 documentation and
analysis system (Alpha Innotech Corp., San Leandro, CA) (Fig.
2). The BGAF-Dhr1 binding assay
was essentially the same as described above, except that mobility
shifts of BGAF were analyzed by immunoblotting using anti-BGAF serum as
probe instead of enzymatic activity, because Dhr1 does not hydrolyze MUGlc. Initially, purified BGAF was incubated in 2-fold incremental concentrations ranging from 20 to 2.5 µg/ml with a fixed volume of a
Dhr1 expression extract. Mixtures were electrophoresed on 8% native
gels, blotted, and probed with anti-BGAF sera (Fig. 3). BGAF by itself and BGAF incubated
with Glu1 served as negative and positive controls, respectively, in
BGAF-Dhr1 binding assays.
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Fig. 2.
Gel-shift assay (8% native PAGE;
anode at bottom) for the detection of
BGAF binding to maize Glu1 and Glu2 (both bind BGAF) and their chimeras
with each other and sorghum -glucosidase Dhr1
(does not to bind BGAF). Purified BGAF (37 pmol; lane
2) was incubated with ~3 pmol of purified Glu1, Glu2, C-2, C-4,
C-16, C-15, C-21, and C-22. In the case of chimeras C-3, C-5, C-6,
C-19, and C-26 crude expression extracts were used to mix with purified
BGAF. Note that
-glucosidase activity zones (bands and
smearing) detected with MUGlc are retarded in a region
extending from the top of the resolving gel to the sample
well in the stacking gel when BGAF binds wild-type
-glucosidases
(lanes 4 and 6) and their chimeras (lanes
8, 10, 12, 14, 16,
18, 20, 22, and 24). In the
case of C-5, there were two distinct
-glucosidase-BGAF complexes,
whereas in others the complexes formed a smeared zone.
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Fig. 3.
Immunoblot (right panel)
after native PAGE showing that the sorghum
-glucosidase Dhr1 does not bind BGAF, showing no
evidence of BGAF removal, whereas Glu1 (left panel,
positive control) binds BGAF, as evident from the absence of BGAF in
lanes 2.5 and 5 µg/ml and significant decrease
in 10 and 20 µg/ml. A fixed amount of Glu1
was incubated with BGAF at 2-fold decreasing increments of BGAF,
electrophoresed on 8% native gels (anode at
bottom), blotted, and probed with anti-BGAF serum.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucosidase interaction and define the location of the
putative binding site(s), chimeric
-glucosidases were constructed
(Fig. 1B). The interaction between BGAF and
-glucosidase
is very specific and reminiscent of an antigen-antibody interaction,
therefore, binding assays could be performed with both purified and
unpurified ligands with no effect on assay sensitivity and specificity
(11). Consequently, 7 (Dhr1, C-3, C-5, C-6, C-18, C-19, and C-26) out
of 16
-glucosidases were used as crude bacterial cell lysates in
binding assays.
-glucosidase complexes with
reduced electrophoretic mobility on native PAGE gels. Thus,
-glucosidase activity zones (bands) shifted toward the cathodic end
of the gel and a smear extending from the top of the resolving gel to
the sample well in the stacking gel was present (Fig. 2, lanes
4, 6, and 20). The binding assays also
demonstrate that when the C-terminal 47 amino acids from either Glu1 or
Glu2 are replaced with the corresponding C-terminal 53 amino acids from Dhr1 (as is the case with C-2 and C-4, respectively) BGAF binding activity is almost completely lost (Fig. 2, lanes 8 and
10). However, when the C-terminal 23 amino acids of the
Glu1/Dhr1 chimera C-2 are replaced by the C-terminal 17 amino acids of
Glu1, which yielded C-16, BGAF binding activity is mostly restored
(Fig. 2, lane 12). Not surprisingly, replacing the extreme
C-terminal 17 amino acids of Glu1 by the 23 amino acids of Dhr1 (C-15)
showed BGAF binding activity (Fig. 2, lane 14). The binding
data from chimeras C-21 and C-22 show that BGAF binding becomes tighter
when the disruptive region (from Dhr1) spanning amino acids
Ser466-Leu495 of C-16 is bisected, yielding
C-21 and C-22 (Fig. 2, lanes 16 and 18). In the
case of Dhr1/Glu1 or Dhr1/Glu2 chimeras, in which the N-terminal Glu1
or Glu2 regions were replaced with the N-terminal Dhr1 regions varying
from 127 (C-26) and 205 (C-19) to 461 amino acids long (C-18, data not
shown), no BGAF binding to any of these three chimeras was observed
(Fig. 2, lanes 26 and 28).
-glucosidase (Glu1) increased,
because the resulting complex was unable to enter the gel (Fig. 3,
left panel). Furthermore, Dhr1 did not bind BGAF in
coprecipitation assays (data not shown) performed as described previously (11).
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Fig. 4.
Three-dimensional structure of the maize
-glucosidase isozyme Glu1 showing a surface patch
formed by amino acids that occur in two different regions (N-terminal
Glu50-Asn127 and C-terminal
Phe466-Ala512). Within the patch
(postulated BGAF binding site), the residues shown in red
are invariant in Glu1, Glu2, and Dhr1, whereas those in
purple are unique to Glu1 (and Glu2) and different in Dhr1.
The Glu1 and Glu2-specific residues (purple) are postulated
to be essential for BGAF binding, whereas one or more of the homologous
Dhr1-specific sites are disruptive for BGAF binding. The variant sites
shown in light blue may not be essential for binding because
of their location or functional equivalence or variation between Glu1
and Glu2. The arrowhead on the left points to the
active site, which is away from the BGAF binding site.
-glucosidase (see below).
The experimentally determined N-terminal sequence VISNKAPIGI of the
mature protein starts 38 amino acids after the first methionine in the
precursor. Thus, the BGAF precursor has a 38-amino acid-long
presequence (i.e. signal peptide), which leaves a mature
protein that is 268 amino acids long after the cleavage of the signal
peptide.
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Fig. 5.
Primary structure of the putative BGAF
precursor protein. The two octapeptide repeat regions postulated
to be involved in binding to maize -glucosidases are shown in
underlined and italicized. The hydrophilicity plot (not shown)
predicts that these regions reside on the surface. The N-terminal
sequence (in boldface) determined by sequencing a purified
BGAF preparation isolated from the "null" maize inbred H95 follows
a 38-amino acid-long signal peptide (underlined). The
peptide sequence APIGIGAT used for designing oligonucleotide sequences
to amplify the original BGAF cDNA is underlined.
-glucosidase (Fig.
6B, lane 4). In contrast, the putative precursor protein encoding BGAF cDNA yielded a protein (306-amino acid-long protein, calculated molecular mass 31.8 kDa) whose electrophoretic mobility was similar to that of BGAF isolated from plant tissue (Fig.
6A, lanes 5-7). The immunoblot analysis of the
protein products of both BGAF cDNAs showed that they had the same
immunoreactivity toward anti-BGAF serum as did BGAF extracted from
maize shoots (Fig. 6A, lanes 3-7). The
immunoblotting data clearly indicated that the presumptive BGAF
cDNAs encode polypeptides that have the same immunoreactivity with
the anti-native BGAF serum as the native BGAF. More unequivocal
evidence that the putative BGAF cDNAs encode BGAF came from
functional assays. The gel-shift assay in Fig. 6B
(lanes 4 and 6) shows that BGAF expressed in
E. coli binds
-glucosidase and retards its
electrophoretic mobility in a manner identical to that obtained with
native BGAF isolated from maize.
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Fig. 6.
Expression of the BGAF cDNA in E. coli. A, SDS-PAGE gel (12%,
anode at bottom) blot of E. coli
lysates probed with anti-BGAF serum. Lanes 1 and
2, total and soluble protein, respectively, from
nonrecombinant pET21a containing E. coli cells (negative
control); 3 and 4, total and soluble protein,
respectively, from cells containing recombinant pET21a with the mature
protein coding BGAF cDNA; 5 and 6, total and
soluble protein, respectively, from cells containing recombinant pET21a
with the precursor protein coding BGAF cDNA; 7, shoot
extract from maize inbred H95 serving as a positive control. Note that
the mature BGAF protein (lanes 3 and 4) produced
in E. coli is smaller in size (nonglycosylated) than that
produced in the maize plant (lane 7). B, BGAF
binding to -glucosidase detected on a native-PAGE gel (8%,
anode at bottom) after incubation with MUGlc.
Lane 1, lysate of E. coli cells containing
nonrecombinant pET21a (negative control); 2, Glu1 (no BGAF);
3, expression extract from the mature protein coding BGAF
clone (no
-glucosidase); 4, Glu1 + lysate from the mature
protein coding BGAF clone; 5, lysate from the putative
precursor protein encoding BGAF clone; 6, same as lane
5, but mixed and incubated with Glu1; 7, Glu1 + lysate
from cells containing nonrecombinant pET21a (no BGAF). Note that only
the lanes containing recombinant mature (lane 4) and
putative precursor BGAF (lane 6) show retarded
-glucosidase activity zones indicating the presence of a functional
BGAF.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucosidase null phenotype previously reported in maize is
due to a specific interaction between the enzyme and an aggregating factor or BGAF (11). The specificity of the interaction is proven by
the fact that BGAF does not bind
-glucosidases from fungi (Trichoderma and Aspergillus) and other plant
sources (e.g. almond, black cherry, sorghum, rice, and
oats). We have cloned and sequenced the cDNA encoding BGAF
(GenBankTM accession number AF232008) and identified BGAF as a member
of the JAp family (16, 17). It is possible that BGAF is also related
functionally to small heat shock proteins, which (28-30) bind
nonnative proteins, preventing their aggregation and maintaining them
in a state competent for ATP-dependent refolding by other chaperones.
-glucosidase system represents a highly specific
protein-protein interaction, providing insights into the molecular mechanism of the interaction based on binding data from Glu1/Dhr1 and
Glu2/Dhr1 chimeric enzymes. The BGAF binding assays with chimeras clearly show that replacement of the C-terminal 47 amino acids of Glu1
with its Dhr1 homolog abolishes binding (Fig. 2, lane 8),
indicating that this region is essential for BGAF recognition and
binding. The results of binding assays with chimeras C-2, C-4, C-16,
C-15, C-21, and C-22 (Fig. 2, lanes 8, 10,
12, 14, 16, and 18) suggest
that both the C-terminal 28-amino acid-long (Phe466-Lys493 in C-16) and the extreme
17-amino acid-long (Lys496-Ala512 in C-15)
regions of Glu1 are individually capable of restoring BGAF binding,
albeit not to the same extent. Thus, the Glu1 region Phe466-Lys493 is compatible with the Dhr1
region Gln492-Asn514 as is the Dhr1 region
Ser462-Arg489 with the Glu1 region
Lys496-Ala512. This result is not surprising,
because sites for most protein-protein interaction interfaces are
confined to a surface patch that is usually composed of more than one
stretch of the same polypeptide chain, which incrementally and
combinatorially contribute to the interaction with the ligand.
Within the region Phe466-Lys493, Glu1 and Glu2
differ from Dhr1 by eight amino acid substitutions where a given site
in Dhr1 is occupied by a different and nonequivalent (except K493R)
residue from that in Glu1 and Glu2 (Fig. 1A). Of these,
F466S/Y466S, A467S, Y473F, and K493R can be ruled out for involvement in BGAF binding, because the first three are buried in the
active site cavity of the enzyme and the K493R substitution is likely
to be equivalent. This narrows down the candidate sites for
contribution to BGAF recognition and binding to N483G, T485E, Y487T,
and E490R. These four amino acid substitutions separating Glu1 and Glu2
from Dhr1 are likely to have significant effects on BGAF recognition
and binding, because they change the bulkiness, hydrophilicity, or
charge of the side chain. Similarly, within the region
Lys496-Ala512, Glu1 and Glu2 differ from Dhr1
by six amino acid substitutions (K496Q, T500G, K502A, P504K, S505N, and
K506N), an internal dipeptide (VE) addition and a terminal tetrapeptide
addition (GQLN), each of which alone or in combination with others may
affect BGAF recognition and binding (Fig. 1A). In short,
both C-terminal regions (Phe466-Lys493 and
Lys496-Ala512) are individually capable of
complementing an N-terminal region (Glu50-Val145, see below) to form a functional
BGAF binding site. The finding that the BGAF binding site on Glu1 and
Glu2 is made up of more than one stretch of polypeptide is also
supported by the finding that chimeras C-18 (data not shown) and C-19
do not bind BGAF (Fig. 2, lane 26). Although the data
indicate that the BGAF binding site is a surface patch that includes
certain amino acids from the C-terminal (e.g. C-2, C-4)
region, they alone are not sufficient to evoke BGAF binding. Moreover,
BGAF showed no binding activity toward inactive, denatured Glu1
extracted from inclusion bodies (data not shown), indicating that the
tertiary structure of the correctly folded enzyme is essential to form
a functional binding site.
-glucosidase tertiary
structure to form a functional BGAF binding site. This provides a
plausible explanation for the lack of BGAF binding to Dhr1 and certain
Glu1/Dhr1 or Glu2/Dhr1 chimeras (C-2, C-4, C-18, C-19, and C-26) in
which local structural changes due to amino acid substitutions at one
or more of the 11 plus postulated sites disrupt binding.
-glucosidase aggregating activity in gel-shift
assays (Fig. 6B, lanes 3-6).
-glucosidase binding site or makes a
major contribution to it. The hydrophilicity plot predicts that both
octapeptide repeats reside on the surface of BGAF and, thus, would be
available for interaction with maize
-glucosidases. Our previous
finding that the BGAF-
-glucosidase aggregates can grow to a size in
excess of 1.5 × 106 Da (11) suggests that both BGAF
and
-glucosidase must be bivalent. If BGAF were monovalent, it could
only bind one
-glucosidase dimer, resulting in a soluble quaternary
association of discrete size (~180-190 kDa), which is not observed.
Densitometric analysis of BGAF and
-glucosidase monomer intensities
after cosedimentation suggests a stoichiometry of about 2 molecules of
-glucosidase (two monomers of a homodimer) to 1 molecule of BGAF
(monomer), consistent with the bivalency of both molecules. Based on
the binding assays and the finding of two octapeptide repeats in the primary structure of BGAF, we propose that one BGAF molecule or monomer
binds as a divalent ligand to two
-glucosidase dimers, linking
-glucosidase dimers in a linear chain in which monomeric BGAF with
two binding sites and dimeric
-glucosidase with two BGAF binding
sites alternate (Fig. 7). In fact, the
-glucosidase-BGAF interaction is similar to antigen-antibody
interactions, having an equivalence point (11). When soluble
-glucosidase and BGAF are present in the correct ratios, optimal
precipitation occurs. In the region of either
-glucosidase or BGAF
excess, only small complexes are formed. There are other examples of
-glucosidase aggregation and
-glucosidase binding proteins in
plants.
-Glucosidases from flax and oat occur in high molecular mass
forms ranging from 245 to 1200 kDa (32-34). Additionally, Falk and
Rask (35) reported two myrosinase (
-thioglucosidase)-binding
proteins (50 and 52 kDa) from rapeseed.
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Fig. 7.
A model for
-glucosidase-BGAF interaction leading to
-glucosidase aggregation and insolubility. The
model is based on BGAF-
-glucosidase binding assays, which have
identified one BGAF binding site per
-glucosidase monomer, two per
homodimer. Additionally, BGAF in its uncomplexed form exists as a
monomer and thus, must minimally be bivalent. The two octapeptide
repeats found in the primary structure of BGAF (Fig. 5; G(P/R)WGGSGG)
is postulated to be involved in
-glucosidase recognition and
binding, and they support the model along with stoichiometric data,
which show a ratio of 2 molecules of
-glucosidase (dimer) per one
molecule of BGAF.
It appears that binding of BGAF to -glucosidase does not affect
enzyme activity and kinetic parameters, suggesting that BGAF binding
neither sterically blocks the active site nor changes the conformation
to alter enzyme activity. This suggestion is corroborated by the
finding that the postulated BGAF binding site (formed by residues in
domains Glu50-Asn127 and
Phe466-Ala512) on
-glucosidase is away from
the active site (Fig. 4). One plausible function of
BGAF-
-glucosidase interaction may be that BGAF plays a protective
role for
-glucosidase, shielding the enzyme from endogenous
proteases or proteases in the secretions of invading pests.
Additionally, the BGAF-
-glucosidase interaction would keep active
-glucosidase at the wound site, preventing the enzyme from diffusing
to other parts of the plant where it has been shown to elicit
deleterious effects (36).
In conclusion, we have shown that maize BGAF is a member of the JAp
family that specifically interacts with -glucosidase. Based on
corroboratory binding and structural data we have identified two
different regions in the primary structure of
-glucosidase, which
form a BGAF binding site on the surface of the enzyme. We have also
isolated the BGAF cDNA, deduced the sequence of its protein
product, identified two octapeptide repeats in the sequence, and
postulated that they form two binding sites each of which binds a
monomeric unit of the
-glucosidase homodimer. We have confirmed the
identity of the BGAF cDNA further by expressing it in E. coli and demonstrating that its recombinant protein products (mature and precursor BGAF) are functionally and immunologically identical to the native BGAF isolated from maize. The finding that BGAF
shares significant amino acid identity (58-61%) with three defense
proteins (GenBankTM AF021258, U43496, and U43497) from barley involved
in systemic acquired resistance and the fact that
-glucosidase also
plays a role in defense suggest that the specific interaction between
BGAF and
-glucosidase has physiological relevance. Future studies
will focus on the precise identification of specific amino acids within
the binding sites of BGAF and
-glucosidase and defining their roles
using site-directed mutagenesis and x-ray crystallography, as well as
understanding the physiological function of the BGAF-
-glucosidase interaction.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. David R. Bevan of the Department of Biochemistry, Virginia Tech, for his help with molecular modeling studies.
![]() |
FOOTNOTES |
---|
* This research was supported by Grant MCB-9906698 from the National Science Foundation, by Jeffress Foundation Grant J-377, and by a Sigma Xi grant in aid of research (to D. J. B.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF232008.
To whom correspondence should be addressed: Tel.: 540-231-8951;
Fax: 540-231-9307; E-mail: aevatan@vt.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008872200
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ABBREVIATIONS |
---|
The abbreviations used are:
BGAF, -glucosidase aggregating factor;
JAp, jasmonic acid-induced protein;
PCR, polymerase chain reaction;
AS, ammonium sulfate;
PAGE, polyacrylamide gel electrophoresis;
MUGlc, 4-methylumbelliferyl-
-D-glucoside;
EST, expressed
sequence tag;
DIMBOAGlc, 2-glucopyranosyl
4-hydroxy-7-methoxy-1,4-benzoxazin-3-one;
DIMBOA, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one.
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REFERENCES |
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