Plant Science 156 2000 55 – 63
Glycosylation of the cationic peanut peroxidase gene expressed in transgenic tobacco
Bao Lige
a
, Shengwu Ma
b
, Robert B. van Huystee
a,
a
Department of Plant Sciences, Uni6ersity of Western Ontario, London, Ont., Canada N
6
A
5
B
7
b
Siebens-Drake Research Institute,
1400
Western Road, London, Ont., Canada N
6
G
2
V
4
Received 19 August 1999; received in revised form 22 February 2000; accepted 22 February 2000
Abstract
The major cationic peanut Arachis hypogaea peroxidase, secreted into the extracellular space, is a glycoprotein with three N-linked glycans polysaccharides which are connected to the peptide backbone at Asn-60, Asn-144 and Asn-185. In this report,
a C-terminal histidine-tagged cationic peanut peroxidase gene was expressed in transgenic tobacco Nicotiana tabacum. Tissue of the transgenic tobacco was cultured in suspension culture and the his-tagged peroxidase was purified in large quantities from
14-day-old suspension culture. The number of glycans, glycosylation sites and the chemical nature of glycan moieties attached to cationic peanut peroxidase expressed in transgenic tobacco were examined. Cationic peanut peroxidase isolated from the above
transgenic tobacco had the identical number of complex glycans, attached at the same glycosylation sites as on cationic peanut peroxidase isolated from peanut suspension culture. Monosaccharide components of these glycans are N-acetylglucosamine
GlcNAc, mannose Man, fucose Fuc, xylose Xyl and galactose Gal, the same sugars as found in native cationic peanut peroxidase. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Peroxidase; Peanut; N-linked glycans; Monosaccharide; Glycosylation; Transgenic tobacco www.elsevier.comlocateplantsci
1. Introduction
Glycosylation is one of the most important co- and post-translational modifications to proteins.
Proteins, including plant proteins can be glycosy- lated at either O- or N-glycan binding sites, as
reviewed [1 – 4]. Many functions have been sug- gested for protein glycosylation, including sig-
nalling for intracellular targeting, protection from proteolytic breakdown, maintenance of protein
stability, configuration and correct folding [5 – 9]. Proteins are direct gene products. Therefore,
their sequences are determined by a template. In glycan biosynthesis, no such template is involved
in the processing of the oligosaccharides. More- over, the biosynthesis of complex N-linked glycans
can be affected by various environmental factors [6]. Therefore, glycoproteins may not always be
similarly glycosylated when comparisons are made between different cell types from the same organ-
ism [6,10,11]. Thus, if a glycoprotein is expressed in foreign host cells, its glycosylation could be very
different from the original glycoprotein [12,13], and these modifications may alter the biological
activity of a protein. In addition, the same oligosaccharide on different proteins may have
quite different properties, depending on the orien- tation with respect to its polypeptide [7]. The
sequence and linkage of each of the monosaccha- ride components and the mature oligosaccharide
structure at a specific glycosylation site are compli-
Abbre6iations
:
ABEE, p-aminobenzoic ethyl ester; CM, car- boxymethylcellulose; cPrx, cationic peanut peroxidase; cPrx-his6, six
histidine-tagged cationic peanut peroxidase; Fuc, fucose; Gal, galac- tose; GlcN, glucosamine; GlcNAc, N-acetylglucosamine; Man, man-
nose; NTA, nitrilotriacetic acid; PVDF membrane, polyvinylidene difluoride membrane; TFA, trifluoroacetic acid; TPCK,
L
-1-tosy- lamido-2-phenylethylchloromethyl ketone; Xyl, xylose.
Corresponding author. Tel.: + 1-519-6792111, ext. 6490; fax: +
1-519-6613935. E-mail address
:
huysteejulian.uwo.ca R.B. van Huystee 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 2 3 3 - 8
cated [14] and many factors may be involved. We are interested in understanding the role of glycosy-
lation in the action and formation of cationic peanut peroxidase cPrx. cPrx, a 40-kDa protein,
is the major secreted isozyme of peanut peroxidases [15]. Like many plant extracellular proteins, cPrx is
also an intensively studied glycoprotein with three N-linked glycans which are connected to the
polypeptide backbone at Asn-60, Asn-144 and Asn- 185 [16]. While the function of heme and calcium
in the enzyme are now known [17], little if anything is known about the role of the glycans [18]. The
glycans belong to the complex type and sugar composition analysis shows that N-acetylglu-
cosamine GlcNAc, mannose Man, fucose Fuc, galactose Gal and xylose Xyl are present in all
three glycans [19], in agreement with its structure [14]. The cPrx is synthesised as a pre-protein con-
taining a 22-amino acid N-terminal signal peptide [20]. When its cDNA was expressed in transgenic
tobacco, the signal peptide of cPrx was able to direct the secretion of cPrx into the extracellular space
[21]. As a first step towards the analysis of the biological role of glycans in the function of cPrx, a
C-terminal histidine-tagged cPrx gene was ex- pressed in transgenic tobacco and purified in large
quantities from the tobacco cell suspension culture. Then, the number of glycans, glycosylation sites and
the chemical nature of carbohydrate moieties at- tached to cPrx expressed in transgenic tobacco were
examined. 2. Materials and methods
2
.
1
. Genetic manipulation and production of transgenic plants
For Agrobacterium-mediated transformation, a binary plasmid was assembled as shown in Fig. 1.
A portion was designed to include the full length of cPrx coding gene sequence with its N-terminal
signal peptide facilitating secretion into extracellu- lar medium, plus the codons for C-terminal six
consecutive histidine residues. The latter was added so as to form a recombinant protein permitting
simple identification and purification. The strategy and procedure for DNA manipulation, analysis of
transgenic expression and localization were com- pleted as described [21].
2
.
2
. Production of transgenic tobacco callus suspension culture
For the establishment of callus suspension cul- ture, leaf discs from young transformed plants
known to be expressing cPrx were cultured on agar plates of callus inducing medium supplemented with
250 mgml of carbenicilin and 100 mgml of kanamycin as described [21]. After 5 – 6 weeks,
suspension cultures were initiated in 250 ml liquid callus inducing medium containing 50 mgml of
kanamycin from the developed calli at 25°C with continuous shaking 120 rpm and maintained for
4 – 6 weeks until uniform dividing cell cultures had been established. Large scale cell cultures were
started by expanding the culture from the continu- ously established cell suspension in 250 ml medium.
The cells were grown in same but antibiotic-free liquid medium at room temperature with continu-
ous shaking 140 rpm, and were routinely subcul- tured every 7 days by adding 250 ml of fresh
medium to 250 ml of 14-day-old cell culture grown in 500 ml medium in an 1000-ml flask.
2
.
3
. Purification of the expressed protein from the spent medium
The 14-day-old spent culture medium was sepa- rated from the tobacco cells by vacuum filtration
and subsequently the macromolecular components in the medium were precipitated in 70 acetone at
4°C overnight. The precipitate was centrifuged at 9000 × g for 15 min, and the pellet was resuspended
in
20 mM
Na-acetate buffer
pH 5.0.
Fig. 1. Schematic representation of the construction part of cPrx-his expression vector. cPrx coding sequence with its
N-terminal signal peptide and the codons for C-terminal histidine-tag was placed under the control of double 35S
promoter with a TEV leader sequence. This construct was then inserted into the binary vector plasmid [21] facilitating
the transfer of the chimeric gene into tobacco cells. NOS-ter, nopaline synthase terminator; NPTII, neomycin phospho-
transferase II gene; TEV leader, leader sequence of tobacco etch virus.
After removing the insolubles by centrifugation at 10 000 × g, the supernatant was brought to
80 saturated ammonium sulphate and cen- trifuged at 13 000 × g for 10 min. The pellet was
resolubilized in 20mM Na-acetate buffer pH 5.0, then dialysed against double distilled water
overnight at 4°C, and the flocculated material which appeared during dialysis was removed by
centrifugation at 13 000 × g [15].
The dialysate was placed on a 2 × 15-cm car- boxymethylcellulose CM BioRad column pre-
equilibrated with 20 mM Na-acetate buffer pH 5.0, and intensively washed with 20 mM Na-ac-
etate buffer pH 5.0 to remove the unbound proteins. Then, a continuous gradient from 20 to
300 mM Na-acetate buffer pH 5.0 was used to elute the bound protein. The CM column was
regenerated with 1 M Na-acetate buffer pH 5.0 and re-equilibrated with 20 mM Na-acetate
buffer pH 5.0 [19].
The eluate from the gradient of 20 – 300 mM was precipitated in 70 acetone at − 20°C. The
pellet from centrifugation at 13 000 × g was air dried and resuspended in 10 mM imidazole pH
7.0. Then, the protein solution was loaded on a 1.5 × 15-cm column of Ni
2 +
-nitrilotriacetic acid NTA resin pre-equilibrated with 10 mM imida-
zole pH 7.0. Prior to elution, the bound mate- rial
was washed
extensively with
30 mM
imidazole pH 6.3 to remove the non-specifically bound proteins. The bound protein was eluted
with 300 mM imidazole pH 5.0 [21]. The eluate was precipitated in 70 acetone at
− 20°C
overnight and the pellet from centrifugation at 13 000 × g was air dried, and resuspended in 1
mM NH
4
-acetate pH 5.0 for analysis. The column was regenerated with 600 mM imidazole
pH 5.0 and re-equilibrated with 10 mM imida- zole pH 7.0. The purity of six histidine-tagged
cationic peanut peroxidase cPrx-his6 was exam- ined by SDS-PAGE [22] and measuring the RZ
value.
2
.
4
. Trypsin digestion and gel filtration chromatography
The purified cPrx-his6 30 mg was treated with acidic acetone 0.5 HCl in pure acetone,
vv to remove its heme component as described [23].
Then the
mixture was
centrifuged at
13 000 × g for 15 min and the pelleted protein was resuspended in 3 ml of 0.1 M NH
4
HCO
3
buffer pH 8.0. If any reddish color remained, the acidic acetone treatment was repeated until
all heme moiety was completely removed. The apoprotein was collected by centrifugation, then
air dried and resuspended in 3 – 4 ml of 0.1 M NH
4
HCO
3
buffer pH 8.0, and digested with
L
- 1-tosylamido-2-phenylethylchloromethyl
ketone- TPCK treated trypsin 2 mgml, trypsin dis-
solved in 0.1 M NH
4
HCO
3
buffer, pH 8.0 sub- strate:enzyme = 30:1, ww [16] at 37°C for 2 h.
Then the tryptic peptides were loaded on a Bio- Gel P-6 column 1.5 × 90 cm, molecular weight
range 1000 – 6000 Da, 50 – 150 mesh pre-equili- brated with 0.1 M NH
4
HCO
3
buffer pH 7.0. The same buffer was also used as a mobile
phase, and the elution was carried out at room temperature, at a flow rate of 8 mlh. Glycopep-
tide-containing fractions were identified using a modified phenol sulphuric acid method and col-
lected. In this method, 50 ml of sample was mixed with 50 ml of 5 phenol in a clear 1.5-ml
microtube, and 400 ml of pure sulphuric acid was added. The color of the reaction solution was
observed 15 min later [24]. The fractions con- taining glycopeptides were then dialysed against
HPLC grade distilled water and lyophilized for further analysis.
2
.
5
. Separation of glycopeptides by re6erse phase HPLC
The lyophilized glycopeptides were resolubi- lized in 500 ml HPLC grade deionized water, and
injected onto a C-18 column Jupiter 250 × 4.60 mm, Phenomenex connected to a HPLC system
gold apparatus programmable solvent module 126, Beckman and a Rheodyne injector with a
500-ml sample loop. Elution was performed at room temperature at a flow rate of 1.2 mlmin
in an acetonitrile gradient for 60 min 0 – 5 min, 0.1 trifluoroacetic acid TFA; 5 – 10 min, 0 –
20 acetonitrile; 10 – 35 min, 20 – 25 acetoni- trile; 35 – 50 min, 25 – 45 acetonitrile; 50 – 60
min, 45 – 100 acetonitrile. The peptide elution profile was detected at 230 nm, and the glycan-
containing fractions were identified using the modified phenol-sulphuric acid method as de-
scribed above. The carbohydrate containing frac- tions were pooled accordingly, dialysed against
HPLC grade distilled water, and lyophilized.
2
.
6
. Identification of glycopeptides by N-terminal sequence analysis
The three glycopeptides separated by HPLC were loaded on SDS-gel electrophoresis, respec-
tively, and then transferred onto polyvinylidene difluoride PVDF membranes BioRad. Follow-
ing staining with Coomassie blue, the peptide bands were cut out, destained in 40 methanol,
and sequenced on an Applied Biosystem Model 475 sequencing apparatus equipped with an on-
line model 120A HPLC for phenylthiohydatoin amino acid identification.
2
.
7
. Labelling of sugars with p-aminobenzoic ethyl ester
ABEE The preparation of ABEE reagent and the
derivatization of sugars were completed as de- scribed [19]. To prepare the stock for ABEE
reagent, 330 mg ABEE and 70 mg sodium cyanoborohydride
were dissolved
in 700
ml methanol, and then 82 ml glacial acetic acid was
added. The stock was stored at 4°C. Before use, the ABEE reagent was warmed to 25°C to dissolve
any crystals formed during storage.
To derivatize standard sugars, 80 ml of ABEE reagent was added to aliquots of 20 ml of different
concentrations of standard sugars containing 800 nmol lactose. Lactose was used as an internal
standard and derivatized along with other sugars. Following a brief vortexing, the mixture was incu-
bated in a heating block at 80°C for 55 min. Then 380 ml HPLC grade distilled water was added to
the reaction vials and mixed by vortexing. Chloro- form 1 ml was added to the mixture and vor-
texed vigorously to extract the free ABEE reagent. The chloroform and aqueous phase were separated
by placing the vials on the bench or by brief centrifugation and the chloroform lower phase
was carefully removed by pipette. The chloroform extraction was repeated one more time and,
finally, the clear aqueous phase containing the sugar derivatives was carefully collected and
filtered for HPLC use.
2
.
8
. Acid hydrolysis of glycoprotein and glycopeptides
In order to analyse the sugar composition of the histidine-tagged cPrx as a whole or the sugar
composition of its individual glycans, cPrx or its glycans
were hydrolysed
to their
individual monosaccharides. Glycoprotein 4 mg or gly-
copeptides 2 mg from the HPLC separation were dissolved in 100 ml of HPLC grade distilled water
in a Pierce Reacti-vial 1 × 3 cm, and 100 ml of 4 M TFA was added to give a final concentration of
2 M TFA. The vial was capped tightly, vortexed briefly and placed in a heating block Fisher Scien-
tific at 100°C for 6 h [25]. The hydrolysate was cooled to room temperature and dried completely
using a vacuum dessicator.
For the derivatization of glycoprotein or gly- copeptide hydrolysate, the hydrolysates obtained
from acid hydrolysis was dissolved in 20 ml of 40 mM lactose, and 80 ml of ABEE reagent was
added. Other procedures were the same as the derivatization of standard sugars as described
above, and the samples containing the sugar derivatives were also analyzed by HPLC.
2
.
9
. Re6erse phase HPLC analysis of the sugar deri6ati6es and construction of calibration cur6es
for quantitati6e analysis The samples of ABEE sugar derivatives in the
aqueous phase were subjected to HPLC C-18 column for sugar analysis using a 20-ml sample
loop. The chromatography was performed at room temperature in an isocratic mode with 86
solvent A 50 mM sodium acetate buffer, pH 4.5 and 14 solvent B 50 mM sodium acetate buffer,
pH 4.5acetonitrilemethanol = 404020, vvv at a flow rate of 2.4 mlmin for 60 min. The sugar
derivatives were detected at 254 nm [19].
To set up standard curves, a range of standard sugars, Man, Gal, GlcNAc, Xyl, Fuc, from 200 to
800 nmol in 20-ml solutions containing 800 nmol of lactose as an internal standard, were derivatized
with ABEE reagent, respectively, and 5 20 ml of the derivatives was analyzed using HPLC as
described above.
3. Results
