Peumans1 1 Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Leuven, Belgium;2Institut de Pharmacologie et Biologie Structurale, Unite´ Mixte de Recherc
Trang 1Purification, characterization, immunolocalization and structural
Els J M Van Damme1, Jialiang Hu1, Annick Barre2, Bettina Hause3, Geert Baggerman4, Pierre Rouge´2and Willy J Peumans1
1 Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Leuven, Belgium;2Institut de Pharmacologie et Biologie Structurale, Unite´ Mixte de Recherche Centre National de la Recherche Scientifique 5089, Toulouse, France;3Institute of Plant Biochemistry, Halle, Germany;4Laboratory of Developmental Physiology and Molecular Biology, Katholieke Universiteit Leuven, Leuven, Belgium
An abundant catalytically active b-amylase (EC 3.2.1.2)
was isolated from resting rhizomes of hedge bindweed
(Calystegia sepium ) Biochemical analysis of the purified
protein, molecular modeling, and cloning of the
correspond-ing gene indicated that this enzyme resembles previously
characterized plant b-amylases with regard to its amino-acid
sequence, molecular structure and catalytic activities
Immunolocalization demonstrated that the b-amylase is exclusively located in the cytoplasm It is suggested that the hedge bindweed rhizome b-amylase is a cytoplasmic vegetative storage protein
Keywords: b-amylase; Calystegia sepium; hedge bindweed; immunolocalization; vegetative storage protein
Exo-hydrolases catalyzing the release of b-maltose from the
nonreducing ends of a-1,4-linked oligo- and polyglucans
(also so-called b- or exo-amylases) (EC 3.2.1.2) have been
studied for several decades because they are possibly
involved in starch metabolism in plants, and play an
important role in biotechnological processes whereby starch
is converted into simple sugars In the past, research on
b-amylases has been focussed on the abundant b-amylases
found in the endosperm of barley (Hordeum vulgare ) and
some other cereals [1], soybean (Glycine max ) seeds [2] and
sweet potato (Ipomoea batatas ) tubers [3] During the last
decade, evidence has accumulated that b-amylases are
ubiquitous in flowering plants Cereals such as barley, wheat
(Triticum aestivum ), rye (Secale cereale ) and maize (Zea
mays ) also contain, besides the classical abundant and
highly active endosperm b-amylases, low levels of another
so-called ‘tissue-ubiquitous’ form in leaves and roots [1]
b-Amylases have also been identified in roots of alfalfa
(Medicago sativa ) and several other forage legumes
including sweetclover (Melilotus officinalis ), red clover
(Trifolium pratense ), birdsfoot trefoil (Lotus corniculatus )
[4], and in pea (Pisum sativum ) epicotyls [5] In addition, b-amylases have been identified in species of the families Solanaceae (potato, Solanum tuberosum ) [6] and Brassica-ceae (Arabidopsis thaliana and Streptanthus tortuosus ) [7,8]
Extensive enzymatic studies of several b-amylases unambiguously demonstrated that these enzymes exclu-sively catalyze the release of b-maltose from the nonreducing ends of a-1,4-linked oligo- and polyglucans Accordingly, b-amylases are believed to be involved in the degradation of starch in the plant and/or a-1,4-linked oligoglucans Though this presumed role might hold true for some b-amylases, it certainly cannot be extrapolated to all plant b-amylases because (a) some b-amylases occur in tissues that are devoid of starch, (b) many plant b-amylases are spatially separated from their presumed substrate (i.e starch), and (c) inbred lines of rye lacking the abundant endosperm b-amylase germinate normally [9] This implies that some b-amylases are not required and even not involved
in starch degradation but fulfil another role [10] It has been proposed, for example, that the abundant b-amylases from cereal endosperm and alfalfa taproots function as seed storage proteins and vegetative storage proteins (VSPs), respectively [1,4] A major difficulty in confirming the role
of b-amylases is the lack of insight in their subcellular location According to some reports, b-amylase is an extrachloroplastic protein restricted to the cytoplasm of spinach cells [11] and A thaliana leaves [7], which implies that the enzyme does not contribute to the amylolytic activity of the chloroplast Others, however, presented evidence for a vacuolar location (e.g in pea and wheat leaf protoplasts) [12] Indirect evidence based on the absence of
a signal peptide from the deduced sequence of all b-amylases cloned thus far suggests that the enzyme is located in the cytoplasm [10] Although there is evidence that in A thaliana leaves one particular b-amylase is
Correspondence to E J M Van Damme, Katholieke Universiteit
Leuven, Laboratory for Phytopathology and Plant Protection, Willem
de Croylaan 42, 3001 Leuven, Belgium Fax: þ 32 16 322976,
Tel.: þ 32 16 322379, E-mail: Els.VanDamme@agr.kuleuven.ac.be
Enzyme: b-amylase (EC 3.2.1.2).
Note: the nucleotide sequence reported in this paper has been submitted
to the GenBanke/EMBL Data library under the accession number
AF284857.
(Received 6 July 2001, revised 5 October 2001, accepted 8 October
2001)
Abbreviations: CalsepRRP, Calystegia sepium RNase-related protein;
HCA, hydrophobic cluster analysis; VSP, vegetative storage protein;
Calsepa, C sepium agglutinin.
Trang 2synthesized with a typical N-terminal chloroplast import
signal and is efficiently imported by isolated pea
chloroplasts [13], it is still unclear whether plant b-amylases
in general are transported from the cytoplasm into another
subcellular compartment
A recent study of the predominant proteins in rhizomes of
hedge bindweed (Calystegia sepium ) revealed that this
vegetative storage tissue accumulates, besides large
quantities of a catalytically inactive RNase-related protein
[14], substantial amounts of a mannose/maltose-specific
lectin [15,16] and a 55-kDa polypeptide with an N-terminal
sequence similar to that of typical plant b-amylases This is
an interesting observation because it demonstrates for the
first time the simultaneous occurrence in a plant tissue of a
lectin with a high affinity for the reaction product of
b-amylases To confirm the possible interaction between the
carbohydrate-binding protein and the
polysaccharide-degrading enzyme the hedge bindweed b-amylase was
purified, characterized and immunolocalized Our results
demonstrate that the enzyme resembles previously
described plant b-amylases and is exclusively located
in the cytoplasm The abundance, subcellular location
and developmental regulation suggest that the rhizome
b-amylase is a cytoplasmic VSP
M A T E R I A L S A N D M E T H O D S
Plant material
Rhizomes of hedge bindweed [C sepium (L.) R.Br.] were
collected in Leuven in winter
Extraction and purification of b-amylase from rhizomes of
C sepium
The b-amylase was purified by classical protein purification
techniques Fresh rhizomes (100 g) were cut into small
pieces and homogenized in a Waring blender in 1 L of a
solution of 0.1% (w/v) ascorbic acid (adjusted to pH 6.5)
The homogenate was squeezed through a double layer of
cheesecloth and centrifuged at 3000 g for 10 min The
supernatant was adjusted to pH 8.7 with 1M NaOH,
centrifuged at 8000 g for 10 min and filtered through filter
paper (Whatmann 3 mm) A first purification step was
achieved by ion-exchange chromatography The crude
extract was applied on a column (5 £ 5 cm; 100 mL bed
volume) of Q Fast Flow (Amersham Pharmacia Biotech,
Uppsala, Sweden) equilibrated with 20 mM Tris/HCl
(pH 8.7) After loading the extract the column was washed
with 1 L of the same Tris buffer and eluted with 300 mL of
0.2M NaCl in Tris buffer The resulting partially purified
protein fraction was diluted with 5 vol of Tris buffer and
loaded on a column (2.6 £ 15 cm; 75 mL bed volume) of
Q Fast Flow (Amersham Pharmacia Biotech, Uppsala,
Sweden) equilibrated with Tris buffer After washing with
200 mL of Tris buffer, proteins were eluted with a linear
gradient (500 mL) of increasing NaCl concentration (from 0
to 0.4M) in Tris buffer Fractions (10 mL each) were
collected and the proteins analysed by SDS/PAGE All
fractions containing predominantly a single polypeptide of
< 55 kDa were pooled, adjusted to 1M ammonium
sulfate (by adding the solid salt) and applied on a
column (1.6 cm £ 5 cm; < 10 mL bed volume) of
phenyl – Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with 1Mammonium sulfate Bound proteins were eluted with 5 mL of 0.1M Tris/HCl (pH 8.7) and loaded onto a column (2.6 £ 70 cm;
< 350 mL bed volume) of Sephacryl 100 equilibrated with KCl/NaCl/Pi (1.5 mM KH2PO4/10 mM Na2HPO4/
3 mM KCl/140 mM NaCl, pH 7.4) The main peak eluting with an apparent molecular mass around 200 kDa was collected, dialysed against appropriate buffers and stored in small aliquots at 2 20 8C until use Analysis by SDS/PAGE confirmed that the purified protein consisted exclusively of a single 55-kDa polypeptide Activity assays demonstrated that the protein exhibited b-amylase activity
Analytical methods Purified proteins were analyzed by SDS/PAGE using 12.5 – 25% (w/v) acrylamide gradient gels as described by Laemmli [17] The gel was scanned with an AlphaImagere
2200 documentation and analysis system (Alpa Innotech Corporation, San Leandro, CA, USA) to determine the relative concentrations of the major proteins
For N-terminal amino-acid sequencing the proteins were separated by SDS/PAGE and electroblotted onto a poly(vinylidene difluoride) membrane Polypeptides were excised from the blots and sequenced on an Applied Biosystems model 477 A protein sequencer interfaced with
an Applied Biosystems model 120 A on-line analyzer Isoelectric focusing was performed on the Pharmacia Phast System using polyacrylamide gels (5% T/3% C) containing ampholytes (pH 3 – 9) (Amersham Pharmacia Biotech) The proteins were detected with silver staining (Pharmacia LKB Biotechnology, Development Technique File no 210) and isoelectric focusing standards (pI 3.5 – 9.3) were used
Total neutral sugar content of the purified protein was determined by the phenol/H2SO4 method [18], with
D-glucose as standard
For alkylation, 1 mg purified protein was dissolved in
200 mL 0.1M Tris/HCl (pH 8.7) containing 8M urea and
10 mM 2-mercaptoethanol After heating at 60 8C for
10 min iodoacetamide was added to a final concentration of
20 mMand the mixture kept on ice for 30 min The reaction was quenched by the addition of 2-mercaptoethanol (50 mM
final concentration) followed by heating at 60 8C for 10 min Mass spectrometry was performed using a MALDI-TOF instrument Two microliters of a 1.3-mg·mL21b-amylase solution were mixed with one microliter of a 50-mMsolution
of a-cyano-4-hydroxycinnamic acid in CH3CN/EtOH/ trifluoroacetic acid (50 : 49.9 : 0.1) and applied on the multi sample target This mixture was air-dried and the target was then introduced in the instrument, a VG Tofspec
SE (Micromass, Manchester, UK) equipped with a N2-laser (337 nm) The samples were measured in the linear mode (acceleration voltage 25 kV), the laser energy was reduced until an optimal resolution and signal/noise ratio was obtained The results of 10 – 20 shots were averaged to obtain the final spectrum
Enzyme assay The b-amylase activity was determined by different methods The first method was based on the release of
Trang 3p-nitrophenol from the specific substrate p-nitrophenyl
maltopentaoside (Betamyl reagent from Megazyme,
Wicklow, Ireland) [19] Assays were performed for
10 min at 40 8C in maleate buffer (pH 6.2), and absorbance
was measured at 410 nm as described in the manual
provided by the manufacturer This method is highly
specific for b-amylases Moreover, it is a simple and
sensitive test but is less suited for kinetic analysis In a
second method the release of maltose residues from starch,
amylopectin and amylose (Sigma Chemical Co., St Louis,
MO, USA) was measured by the dinitrosalicylic acid
method [20] Enzyme solution (0.2 mL) and 0.2 mL
substrate (0.0625 – 1% starch, amylopectin or amylose)
were incubated for 15 s to 3 min at 20 8C in buffer (pH 5)
The reaction was stopped by adding 0.4 mL of staining
solution [1% dinitrosalicylic acid (w/v) and 30% sodium
potassium tartrate (w/v) in 0.4M NaOH] and heating at
90 8C for 5 min before measuring the absorbance at 540 nm
against blanks without enzyme The dinitrosalicylic acid
method is not very specific for b-amylase in that it will also
measure a-amylase activity Moreover, it is time consuming
and relatively insensitive However, the method is very well
suited for kinetic analyses of purified b-amylases In a third
method the degradation of starch was determined by the
iodine staining method [21] The reaction was started by
adding 100 mL of the enzyme (21.4 mg·mL21) to 500 mL
substrate solution [0.0625 – 1% (w/v)] and 400 mL inhibitor
solution (0.2Mglucose or maltose, or 3.125 mM
cyclohexa-amylose), in 20 mM sodium-acetate buffer (pH 5.0) After
incubation at 20 8C for 15 s to 2.5 min the reaction was
stopped by adding 0.5 mL 1MHCl To each sample 1 mL
staining solution (0.2% iodine in 2% potassium iodide) was
added, and the mixture diluted to 20 mL before measuring
the decrease in absorbance at 700 nm The iodine staining
method also is not very specific for b-amylases and is
relatively insensitive However, the method is less time
consuming than the dinitrosalicylic acid method and is not
affected by the inhibitors of the enzymatic activity
Therefore the iodine staining method is well suited for
extensive kinetic analyses of purified b-amylases
Stability tests
The heat stability of the enzyme was determined by heating
a solution of the purified protein (0.1 mg·mL21 in 0.1M
phosphate buffer pH 6.2) at 20 – 100 8C (with 10 8C
increments) for 10 min Afterwards, activity of the enzyme
was determined using the Betamyl b-amylase test reagent
(Megazyme, Wicklow, Ireland)
To determine the pH stability of the b-amylase, aliquots
of a solution of the purified protein (4.06 mg·mL21in water)
were adjusted to different pH values in a range between 2
and 12, and incubated for 1 h at 25 8C Then 0.1 vol of a
solution of 0.5Msodium acetate (pH 5.0) was added and the
activity of the enzyme was measured by the dinitrosalicylic
acid method
Inhibition of the enzyme activity by glucose, maltose and
cyclohexaamylose
For the study of the enzyme inhibition by glucose, maltose
and cyclohexaamylose b-amylase activity was measured
using the iodine staining method with soluble starch as a
substrate [21] The inhibition type of glucose, maltose and cyclohexaamylose was determined from Lineweaver – Burk plots, and inhibitor constants were determined from Dixon plots
RNA isolation, construction and screening of cDNA library Total cellular RNA was prepared from the apexes of bindweed rhizomes and poly(A)-rich RNA enriched by chromatography on oligo-deoxythymidine cellulose as described by Van Damme and Peumans [22] A cDNA library was constructed as described previously [20] Recombinant clones encoding b-amylase were screened using 32P-end-labeled degenerate oligonucleotide probes derived from the N-terminal amino-acid sequence of Calystegia b-amylase In a later stage, cDNA clones encoding b-amylase were used as probes to screen for more cDNA clones Hybridizations were performed overnight as reported previously [15] Colonies that produced positive signals were selected and rescreened at low density using the same conditions Plasmids were isolated from purified single colonies on a miniprep scale using the alkaline lysis method as described by Mierendorf and Pfeffer [23] and sequenced by the dideoxy method [24] DNA sequences were analysed using programs from PC GENE (Intelli-genetics, Mountain View, CA, USA) and GENEPRO
(Riverside Scientific, Seattle, USA)
Northern blot analysis RNA electrophoresis was performed according to Maniatis
et al [25] Approximately 3 mg of poly(A)-rich RNA were denatured in glyoxal and dimethylsulfoxide and separated in
a 1.2% (w/v) agarose gel Following electrophoresis the RNA was transferred to Immobilon N membranes (Millipore, Bedford, USA) and the blot hybridized using a random-primer-labeled cDNA insert or an oligonucleotide probe Hybridization was performed as reported by Van Damme et al [26] An RNA ladder (0.16 – 1.77 kb) was used as a marker
PCR amplification of genomic DNA fragments encoding b-amylase
DNA was extracted from young leaves of C sepium using the protocol described by Stewart and Via [27] The DNA preparation was treated with RNase (Roche Diagnostics GmbH, Mannheim, Germany) The reaction mixture for amplification of genomic DNA sequences contained 10 mM
Tris/HCl, pH 8.3, 50 mMKCl, 1.5 mMMgCl2, 100 mg·L21 gelatin, 0.4 mM of each dNTP, 2.5 U of Taq polymerase (Roche Molecular Biochemicals, Mannheim, Germany),
500 ng of genomic DNA and 20 mL of the appropriate primer mixtures (20 mM), in a 100-mL reaction volume The reaction was overlaid with 80 mL of mineral oil After denaturation of the DNA for 5 min at 95 8C amplification was performed for 30 cycles through a regime of 1 min template denaturation at 92 8C followed by 1 min primer annealing at 55 8C and 3 min primer extension at 72 8C using a Perkin Elmer DNA Thermal Cycler (480) The PCR fragments were purified using Qiaquick PCR Purification kit (Qiagen, Hilden, Germany) and cloned in TOPO
Trang 4pCR2.1-TOPO cloning vector using the TOPO cloning kit
from Invitrogen (Carlsbad, CA, USA)
Preparation of specific antibodies against b-amylase and
C sepium RNase-related protein (CalsepRRP)
Polyclonal antibodies were raised against b-amylase and the
unglycosylated isoform of CalsepRRP [14] Male New
Zealand white rabbits were injected with 1 mg purified
protein dissolved in KCl/NaCl/Piand emulsified in 1 mL of
Freund’s complete adjuvant Five booster injections with
1 mg of purified protein in 1 mL of KCl/NaCl/Piwere given
at 10-day intervals Ten days after the final injection, blood
was collected from an ear marginal vein After clotting, the
crude serum was prepared by centrifugation (3000 g for
5 min) and processed immediately by affinity
chromato-graphy on a column of immobilized b-amylase or
CalsepRRP Coupling of the antigens to the column and
purification of the antiserum were performed as described
previously [28]
Western blot analysis
The specificity of the antisera was analysed by Western blot
analysis Proteins were separated by SDS/PAGE and
electroblotted on an Immobilon P membrane Before
immunodetection the free binding sites on the membrane
were blocked with 5% BSA in Tris/NaCl/Pi (10 mM Tris,
150 mMNaCl, 0.1% Triton X-100, pH 7.6) for 1 h at room
temperature After washing the membrane with Tris/NaCl/Pi
for 5 min the membrane was consecutively treated with
rabbit primary antibody (overnight incubation at room
temperature), goat anti-(rabbit IgG) Ig (1 h incubation at
room temperature) and peroxidase – anti-peroxidase
com-plex (1 h incubation at room temperature) After every
treatment the membrane was washed three times with
Tris/NaCl/Pi for 5 min Prior to the immunodetection the
membrane was washed for 5 min with 0.1M Tris/HCl,
pH 7.6 The peroxidase reaction was carried out in a fresh
solution of 0.1M Tris/HCl pH 7.6 containing 0.7 mM3,30
-diaminobenzidine tetrahydrochloride and 0.01% (v/v)
H2O2 The reaction was stopped by washing the membrane
in distilled water
Immunocytochemistry
Small pieces of fresh C sepium rhizomes were fixed with
4% paraformaldehyde/0.1% Triton X-100 in KCl/NaCl/Pi,
embedded in poly(ethylene glycol) and cut as described
previously [29] Cross-sections (2 mm thick) were
immuno-labeled by incubation with purified primary IgG raised
against b-amylase or CalsepRRP (diluted 1 : 250 in KCl/
NaCl/Pi containing 5% BSA and 1 mg·mL21 goat IgG)
followed by goat anti-(rabbit IgG) Ig conjugated with
BODIPY (Molecular Probes, Eugene, OR) After
immuno-labeling, sections were mounted in citifluor/glycerol
Control experiments were performed by omitting the
primary antibody The fluorescence of immunolabeled
b-amylase and CalsepRRP was visualized with a Zeiss
Axioskop epifluorescence microscope using the appropriate
filter combination Micrographs were taken by a CCD
camera (Sony, Japan) and processed through thePHOTOSHOP
program (Adobe, Seattle, WA, USA)
Molecular modelling Multiple amino-acid sequence alignments based on
CLUSTAL W[30] were performed withSEQPUP(D.G Gilbert, Biology Department, Indiana University, Bloomington, IN, USA) The program SEQVU (J Gardner, The Garvan Institute of Medical Research, Sydney, Australia) was used
to compare the amino-acid sequences of the b-amylases Hydrophobic cluster analysis (HCA) [31,32] was performed to delineate the structurally conserved b sheets and a helices along the amino-acid sequences of the b-amylase from hedge bindweed and the model b-amylase from soybean HCA plots were generated using the program HCA-Plot2 (Doriane, Paris, France)
Molecular modeling of the b-amylase from C sepium was carried out on a Silicon Graphics O2 R10000 workstation, using the programs INSIGHTII, HOMOLOGY AND DISCOVER (Molecular Simulations, San Diego CA, USA) The atomic coordinates of the soybean b-amylase (code 1BYA) [33] were taken from the RCSB Protein Data Bank (http://www.rcsb.org/pdb) to build the three-dimensional model of the C sepium b-amylase Energy minimization and relaxation of the loop regions was carried out by several cycles of steepest descent and conjugate gradient using the CVFF forcefield of DISCOVER Steric conflicts resulting from the replacement or the deletion of some residues in the C sepium b-amylase were corrected during the model building procedure using the rotamer library [34] and the search algorithm implemented in the
HOMOLOGY program [35] to maintain proper side chain orientation The program TURBOFRODO (Bio-Graphics, Marseille, France) was used to calculate the Ramachandran plot and perform the superposition of the models Electrostatic potentials were calculated and displayed with
GRASPusing thePARSE3 parameters [36] The solvent probe radius used for molecular surfaces was 1.4 A˚ and a standard 2.0 A˚ Stern layer was used to exclude ions from the molecular surface [37] The inner and outer dielectric constants applied to the protein and the solvent were, respectively, fixed at 4.0 and 80.0, and the calculations were performed keeping a salt concentration of 0.14MNaCl No even distribution of the net negative charge of the carboxylic group of negatively charged residues was performed between their two oxygen atoms prior to the calculations The surfaces occupied by negatively charged (Asp, Glu) residues on the solvent accessible surfaces of the modelled amylase were calculated using theGRASPfacilities
R E S U L T S
Purification and partial characterization of the b-amylase fromC sepium rhizomes
SDS/PAGE of clarified homogenates from resting hedge bindweed rhizomes revealed several major polypeptides (Fig 1A), some of which have been identified previously The 15-kDa polypeptide (< 30% of the total protein) corresponds to the subunits of the mannose-binding
C sepium agglutinin (also called Calsepa) [15,16] whereas the 26 to 28-kDa polypeptides (together < 35% of the total protein) represent the unglycosylated and glycosylated form
of the so-called CalsepRRP [14] N-terminal sequencing of the 55-kDa polypeptide (< 10% of the total protein) yielded
Trang 5a single sequence APIPGVMPMGNYVPVYVMLP with a
high degree of identity (85%) to the N-terminus of the
b-amylase from sweet potato (Ipomoea batatas ) Therefore
this polypeptide was tentatively identified as a b-amylase
Subsequently the 55-kDa C sepium protein was isolated
and tested for b-amylase activity
The C sepium b-amylase was purified using a combi-nation of conventional protein purification techniques Analysis of a reduced sample of the final preparation by SDS/PAGE yielded a single polypeptide band of < 55 kDa (Fig 1A) The unreduced protein also yielded a major band
of 55 kDa but exhibited an additional minor band of slightly higher molecular mass (< 65 kDa) To check the possible presence of two different polypeptides the protein was analyzed by mass spectrometry Thereby, a single peak of
56 068 Da was detected with no sign for the presence of a higher Mrform No minor band of 65 kDa could be detected
in an alkylated sample of the protein (Fig 1A), which indicates that this 65-kDa polypeptide appearing in the electropherogram of the unreduced protein is an artifact due
to the formation of an intramolecular disulfide bridge after unfolding of the polypeptide in the presence of SDS Native
C sepium b-amylase eluted as a symmetrical peak with an apparent Mrof < 200 kDa upon gel filtration chromatog-raphy on a Superose 12 column (results not shown), indicating that it is a homotetrameric protein Isoelectric focusing of the purified b-amylase yielded a single band with an isoelectric point of < 4.8 (Fig 1B) No carbo-hydrate could be detected in the pure protein using the phenol/sulfuric acid method suggesting that the C sepium b-amylase is not glycosylated
Fig 2 Lineweaver – Burk plots (A) Lineweaver – Burk plots of the activity of C sepium b-amylase on starch, amylose and amylopectin The activity was assayed in 20 m M , pH 5.0 acetate buffer at 20 8C using iodine staining method The enzyme concentration for activity tests on starch, amylose and amylopectin was 2.588 mg·mL 21 , 2.143 mg·mL 21
and 2.679 mg·mL21, respectively The substrate concentration ranged between 0.025 and 0.5% (w/v) (B) Lineweaver – Burk plots of the inhibition of C sepium b-amylase by glucose, maltose and cyclo-hexaamylose The activity was assayed using soluble starch as substrate
in 20 m M , acetate buffer pH 5.0 at 20 8C The concentration of enzyme was 2.14 mg·mL 21 Concentrations of glucose, maltose and cyclo-hexaamylose were 80 m M , 80 m M and 1.25 m M , respectively The substrate concentration ranged between 0.025 and 0.5% (w/v) Fig 1 SDS/PAGE and isoelectric focusing (A) SDS/PAGE of a
clarified homogenate from C sepium rhizomes and purified b-amylase.
Samples were loaded as follows: lane 1, 100 mL total extract from
Calystegia rhizomes; lanes 2 – 5, 20 mg purified b-amylase The major
protein bands in crude extract (lane 1) represent the b-amylase (A), the
glycosylated and unglycosylated RNase-related protein (R) and
the lectin Calsepa (L) Protein samples in lanes 3 and 5 were alkylated.
The samples in lanes 1 – 3 were treated with b-mercaptoethanol; the
protein in lanes 4 – 5 was not reduced Molecular mass reference
proteins (lane R) were lysozyme (14 kDa), soybean trypsin inhibitor
(20 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), bovine
serum albumin (67 kDa) and phosphorylase b (94 kDa) (B) Isoelectric
focusing of b-amylase from C sepium rhizomes Samples were loaded
as follows: Lane 1, purified b-amylase from C sepium rhizomes and
lane 2, soybean trypsin inhibitor (pI, 4.55) pI markers (lane M) were
amylglucosidase (3.50), trypsin inhibitor (4.55), b-lactoglobulin A
(5.20), carbonic anhydrase B (bovine) (5.85), carbonic anhydrase B
(human) (6.55), myoglobulin (acidic band) (6.85), myoglobulin (basic
band) (7.35), lentil lectin (acidic) (8.15), lentil lectin (middle) (8.45)
lentil lectin (basic) (8.65) and trypsinogen (9.30).
Trang 6Enzymatic activity of b-amylase from hedge bindweed
To check the enzymatic activity of the presumed b-amylase
from C sepium rhizomes three different methods were used
The dinitrosalicylic acid method and iodine staining
method, revealed that the purified protein shows activity
towards starch, amylopectin and amylose (Fig 2A), starch
and amylopectin being a better substrate than amylose As
these two methods are not specific for b-amylase (but also
detect a-amylase activity) additional assays were performed
with the Betamyl b-amylase test reagent from Megazyme
(which uses p-nitrophenyl maltopentaoside as a substrate
and is highly specific for plant b-amylases) In this assay the
protein exhibited a high activity, which implies that the
purified C sepium protein is an active b-amylase
The C sepium b-amylase was active in a pH range from 3
to 7 with an optimum near pH 4.8 Stability tests indicated
that the enzyme was heat stable up to 60 8C but was
completely inactivated upon heating for 10 min at 70 8C
The enzyme is stable in a pH range between 3 and 11
Incubation at pH values below 3 and above 12 irreversibly
inactivated the protein Hydrolysis of the Betamyl
b-amylase test reagent from Megazyme was strongly
inhibited by glucose and maltose At a concentration of
125 mM both sugars reduced the activity of the b-amylase
with 87.5% Mannose caused a 6% reduction of the activity
when added at a final concentration of 125 mM In contrast,
lactose did not inhibit the enzyme even when the
concentration was increased to 250 mM The inhibition of
the enzyme activity by glucose, maltose and
cyclohexa-amylose was studied in more detail using the iodine staining
method Glucose behaved as a mixed type inhibitor whereas
maltose and cyclohexaamylose behaved as competitive
inhibitors of the Calystegia b-amylase (Fig 2B) Glucose
was only a weak inhibitor (Ki ¼ 262 mM) when compared
to maltose (Ki ¼ 11.7 mM) and cyclohexaamylose
(Ki ¼ 0.36 mM)
Molecular cloning of theC sepium b-amylase
Screening of a cDNA library constructed with poly(A)-rich
RNA from rhizome apexes using a synthetic oligonucleotide
derived from the amino-acid sequence of the C sepium
b-amylase yielded multiple positive clones of < 2 Kb
Sequence analysis of Calsepam1 revealed that this clone
contains an ORF of 499 amino acids with one putative
initiation codon at position 2 of the deduced amino-acid
sequence (Fig 3) Translation starting with this methionine
residue results in a protein of 498 amino acids with a
calculated molecular mass of 56 204 Da The deduced
amino-acid sequence of Calsepam1 revealed a sequence
identical to the N-terminal sequence of the protein (residues
A2 – P23) preceded by a methionine residue, suggesting that
the N-terminal methionine residue is removed from the
primary translation product The apparent lack of a signal
peptide further suggests that the b-amylase is localized in
the cytoplasm Removal of the methionine residue results in
a protein of 497 amino acids with a calculated molecular
mass of 56 073 Da and an isoelectric point of 4.81 A search
in GenBank revealed 86% and 67% sequence identity
between the C sepium b-amylase and the b-amylase from
sweet potato and soybean, respectively
Northern blot analysis Northern blot analysis was performed to determine the total length of the mRNA encoding the b-amylase Hybridization
of the blot using the random primer labeled cDNA clone encoding C sepium b-amylase yielded one band of
< 2.0 Kb (results not shown) and is consistent with the length of the cDNA clones which were analyzed
Analysis of genomic fragments encoding b-amylase PCR amplification of genomic DNA fragments encoding b-amylase yielded PCR products of < 2800 bp Sequence analysis revealed a sequence identical to the sequence of the cDNA but split into seven exons by six intron sequences (Fig 3) All introns were marked by GT and AG dinucleotides at their 50 and 30 boundaries, respectively, and were inserted between the third letter of one codon and the first letter of the following codon
Molecular modeling of theC sepium b-amylase The amino-acid sequence of the b-amylase from C sepium exhibits 67.5% identity and 76.0% similarity, respectively, with soybean b-amylase (Fig 3) As the HCA plots of both proteins are very similar, the structurally conserved regions (a helices and b sheets) are readily recognized (results not shown) Due to these structural homologies, a fairly accurate three-dimensional model could be built for the b-amylase from C sepium using the X-ray coordinates of the soybean b-amylase (Fig 4) According to the Ramachandran plot of this model the f and c angles of most of the residues are in the allowed regions of low energy, except for Arg421 (f, 888; c, 1318) It should be mentioned, however, that in the soybean b-amylase also this residue is located in a disallowed region As shown in Fig 4, the model of the b-amylase from C sepium comprises (a) a core built up of a bundle of eight parallel b strands surrounded by eight a helices and thus exhibits a typical (a/b)8barrel structure, (b)
a smaller globular region consisting of three long loops (connected to the core), and (c) a C-terminus consisting of a long loop of over 50 amino-acid residues with a seven amino-acid residue a-helix (Fig 4) The C sepium b-amylase contains six cysteine residues According to the model no disulfide bridges can be formed because the cysteine residues are too distant from each other Only four
of the cysteine residues of the C sepium b-amylase (Cys83, Cys96, Cys209 and Cys344) are homologous to those found
in soybean b-amylase (Cys82, Cys97, Cys208 and Cys343)
On the analogy of the soybean b-amylase, the active site of the C sepium b-amylase most probably consists
of a cleft located between the barrel core and the smaller globular region (Fig 5) This cleft is centered around glutamic residue Glu187 (Glu188 in soybean b-amylase), which is presumed to be involved in the catalytic activity
Localization of the b-amylase in the cells of the rhizomes The cellular and subcellular localization of the C sepium b-amylase was studied by an immunocytological technique Rhizomes embedded in poly(ethylene glycol) were sectioned and immunolabelled with purified antibodies
Trang 7raised against b-amylase The specificity of the
affinity-purified antibodies against the C sepium b-amylase was
checked by Western blot analysis of a crude rhizome extract
As the antibodies reacted exclusively with the 55-kDa
polypeptide corresponding to the b-amylase subunit (results
not shown), they can be considered specific
The b-amylase could be detected in the cortex and the pith of rhizomes but not in vascular tissues, pericycle, endodermis and rhizodermis (Fig 6) In cross-section of cells of the pith and the cortex, the vacuole is the dominant organelle The cytoplasm is visible only as a thin layer adjacent to the cell wall As shown in Fig 6, the b-amylase
Fig 3 Amino-acid sequences (A) Deduced
amino-acid sequence of the b-amylase from
C sepium As the methionine at position 2 is the
first amino acid the residue preceding this
methionine is shown in lower case The sequence
corresponding to the N-terminal sequence of the
protein is underlined The arrowheads indicate the
positions of the intron sequences (B) Comparison
of the amino-acid sequences of b-amylase from
C sepium (this work) with those from Glycine max
(GenBank accession No BAA09462), A thaliana
(accession no BAA07842), Ipomoea batatas
(accession no BAA02286), Triticum aestivum
(accession No P93594), Zea mays (accession no.
P55005) and Hordeum vulgare (accession no.
BAA04815) Please note that the last
17-amino-acid residues at the C-terminus of H vulgare
b-amylase are not shown Deletions are indicated
by dashes and identical residues are boxed.
Trang 8is confined to the cytoplasm (Figs 6B,D) No label for
anti-(b-amylase) IgG was detectable within the large vacuoles,
which appeared as a dark area in the centre of the cells To
clearly distinguish the cytoplasmic location of the C sepium
b-amylase from that of a noncytoplasmic protein, sections
were also immunolabeled with antibodies raised against the
major rhizome protein CalsepRRP, which is presumably
located in the vacuole [14] The specificity of the
affinity-purified antibodies against CalsepRRP was checked by
Western blot analysis of a crude rhizome extract As the
antibodies reacted with the 28- and 26-kDa polypeptides
(corresponding to the glycosylated and unglycosylated
polypeptides of the RNase related protein) (results not
shown), they can be considered specific The results shown
in Fig 6F confirm the vacuolar location of this
RNase-related protein and rule out the possibility that the apparent
cytoplasmic location of the b-amylase is due to an artefact
D I S C U S S I O N
A major protein of resting rhizomes of C sepium, which
accounts for < 10% of the total protein, has been identified
as a b-amylase The native enzyme is a homotetramer of four identical subunits of 56 068 Da It is important to note
in this context that apart from the b-amylase from sweet potato tubers all other plant b-amylases characterized to date have been described as monomeric proteins (which implies that both monomeric and tetrameric b-amylases are catalytically active) Molecular cloning combined with N-terminal sequencing and MALDI-TOF mass spec-trometry indicate that the mature protein comprises the entire open reading frame of the corresponding gene minus the N-terminal methionine residue, which indicates that the
C sepium b-amylase undergoes, apart from the removal of the N-terminal methionine, no co- or post-translational processing According to the results of previously reported molecular and structural studies the processing of the b-amylases from sweet potato [3,38] and soybean [39,40] also is restricted to the removal of the N-terminal methionine whereas that of a phloem-specific b-amylase from A thaliana includes the removal of an N-terminal tetrapeptide [8,41] Cereal b-amylases also are not co- or post-translationally modified [42] However, the abundant endosperm-specific cereal b-amylases are ‘activated and released’ during germination by the proteolytic removal of a C-terminal peptide of < 50 amino-acid residues [1,43] The three-dimensional model of the C sepium b-amylase strongly resembles that of the soybean [33,39] and sweet potato [38] b-amylases and shares the typical (a/b)8barrel core which is common to all other b-amylases of different
Fig 4 Three-dimensional model of Calystegia sepium b-amylase
showing the central bundle of eight strands of b sheet (pink
coloured arrows, numbered 1 – 8) surrounded by eight a-helices
(coloured green, numbered 1 – 8) The a helix in violet does not
participate in the core (b/a) 8 TIM-barrel structure The three acidic
residues involved in the catalytic activity (Asp102, Glu187 and Glu381)
are represented in ball-and-stick The conserved loop 97 – 104
(homologous to loop L3 of the soybean b-amylase), which allows the
active site to close is coloured cyan (H) N and C refer to the N- and
C-terminus of the b-amylase sequence Cartoon was generated with
MOLSCRIPT [51], BOBSCRIPT [52] and RASTER 3 D [53].
Fig 5 Molecular surface of the modelled C sepium b-amylase showing the surface area (black) occupied by the three acidic residues Asp102 (red), Glu187 (blue) and Glu381 (green) located in the central cleft of the (b/a) 8 TIM-barrel Most of the exposed surface of residue Asp102 is poorly visible as it is located inside the cleft whose entry appears as a hole in the centre of the model The model is similarly oriented as in Fig 4 All the calculations and displays were performed with GRASP [36].
Trang 9origins [44] According to both structural [33,39] and
functional data [45 – 47], four amino-acid residues (Asp101,
Glu186, Glu345 and Glu380) located in a cleft occurring
between the (a/b)8 barrel core and the smaller globular
region play a key role in the catalytic activity of the soybean
b-amylase These four residues are conserved in all other
plant b-amylases including the C sepium b-amylase
Studies of the crystal structure of recombinant soybean
b-amylase complexed to b-cyclodextrin demonstrated the
role of Glu186 and Glu380 as catalytic residues [40] It is
important to note in this respect that the distance of 7.89 A˚
between Glu183 (homologous to Glu186 of soybean
b-amylase) and Glu381 (homologous to Glu380 of soybean
b-amylase) of the C sepium b-amylase, fits the inverting
hydrolytic mechanism of b-amylases Binding of maltose or
maltal ligands to the active site of soybean b-amylase
induces a local conformational change of a loop segment
(L3) of eight residues (96 – 103) located in the smaller
globular region This conformational change is required to
close the active site of the enzyme [40,48] and allow the
reaction to take place Once the reaction is finished a new
conformational change is required to bring the loop into the
open position for subsequent release of the reaction product
Due to the importance of the conformational changes the
loop segment L3 is highly conserved in all b-amylases from
plants and microorganisms (e.g
97Gly-Gly-Asn-Val-Gly-Asp-Ala-Val104 of Calystegia b-amylase and
96Gly-Gly-Asn-Val-Gly-Asp-Ile-Val103 of the soybean b-amylase)
Docking experiments with maltose and maltose derivatives
further suggested that the movement of this mobile flap
significantly increased the intermolecular binding potential
and thus favours the interaction with the ligand [49]
Immunolocalization studies of the C sepium b-amylase
provided for the first time unequivocal evidence for the
exclusive cytoplasmic location of a plant b-amylase Our results confirm the presumed cytoplasmic location b-amy-lases proposed on the basis of cell fractionation studies with spinach [11] and Arabidopsis [7] leaves but can not be reconciled with the previously proposed vacuolar location of b-amylase in pea and wheat leaf protoplasts [12] In contrast
to the cytoplasmic b-amylase, the major storage protein of the hedge bindweed rhizome (CalsepRRP) [14] is clearly located in the vacuole This particular vacuolar location of CalsepRRP not only serves as a good endogenous control for the cytoplasmic location of the C sepium b-amylase but also demonstrates that cells of C sepium rhizomes accumulate large quantities of proteins in both the vacuole and the cytoplasm
The cytoplasmic location of the C sepium b-amylase implies that the enzyme has no access to its natural substrate because maltodextrins are believed to accumulate in the vacuole [5] So the question remains why hedge bindweed rhizomes accumulate large quantities of an enzyme with no apparent function This question applies also to all other abundant plant b-amylases, to which for various reasons no clear role can be attributed It has been suggested at several occasions that highly expressed b-amylases may act as storage proteins For example, the absence of a specific function and storage protein-like behaviour of the abundant cereal endosperm b-amylases [1] combined with the observation that mutant lines of barley and rye, which lack the endosperm b-amylase, germinate normally [9,43] point towards a storage role A similar role has been proposed for the abundant b-amylase in taproots of alfalfa, which is believed to fulfil a storage function in the roots of this perennial legume and accordingly is considered a typical VSP [4] The abundance and apparent lack of a specific function suggest that the C sepium b-amylase also can be
Fig 6 Immunolocalization of the C sepium b-amylase in rhizomes of C sepium Cross sections were labelled with a purified polyclonal antibody raised against the C sepium b-amylase (A, B, D) or a purified polyclonal antibody raised against CalsepRRP (F) followed by a fluorescence labelled secondary antibody Immunodecorated b-amylase and CalsepRRP are visible by the green fluorescence (A) Overview of a cross section of a rhizome labelled with anti-(b-amylase) IgG Note the label restricted to the cortex and the pith whereas vascular tissues, pericycle, endodermis and rhizodermis do not exhibit label (B) Detail of (A) showing part of the cortex All cortex cells are labelled (C) Section concomitant to the section shown in (B) without treatment with the first antibodies The weak fluorescence in the vascular tissues is due to the autofluorescence of cell walls containing phenolic compounds (A) concomitant section to (B) is shown (D) Enlargement of (B) to visualize the subcellular localization of the Calystegia sepium b-amylase within cortex cells Note the label clearly visible within the thin cytoplasmic seam only Starch grains of amyloplasts appear as black dots (E) DIC image of (D) to visualize starch grains (F) Cortex cells of a cross section of a rhizome immunolabelled with anti-(CalsepRRP) IgG Cells exhibit strong label within the vacuole Bars represent 100 mm in A – C and 50 mm in D – F, respectively.
Trang 10classified as a VSP (even though the C sepium rhizome can
not be considered a true perennial tissue because it
continuously grows at the one end and dies at the other
end) If so, the C sepium and alfalfa taproot b-amylases
represent a unique type of VSP because they are located in
the cytoplasm whereas all other known VSP are (presumed)
vacuolar storage proteins [50]
A C K N O W L E D G E M E N T S
This work was supported in part by grants from the Research Fund
K.U.Leuven (OT/98/17), CNRS and the Conseil re´gional de
Midi-Pyre´ne´es (A B., P R.), and the Fund for Scientific Research-Flanders
(FWO grant G.0223.97) E V D is a Postdoctoral Fellow of this fund.
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