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
3
.
1
. Purification of the histidine-tagged cPrx from the tobacco medium
The recombinant cPrx secreted by transgenic tobacco was enzymatically active and the intro-
Fig. 2. Verification of the cPrx purification from suspension culture medium of transgenic tobacco cells. After sequential
acetone and ammonium sulphate precipitation, and CM and Ni
2 +
-NTA chromatography, various amounts of purified protein
were visualised
on SDS-PAGE
stained with
Coomassie blue: lane 1, 5 mg; lane 2, 10 mg; lane 3, 15 mg; lane 4, 20 mg; lane 5, 25 mg.
described in [21]. Cell-culture makes the large scale isolation of the expressed protein with high purity
easy and effective. Large amounts of glycoprotein were needed to identify the number, the glycan
binding sites, and the sugar composition. Follow- ing the steps described in Section 2, the result of
purification was visualized on SDS-PAGE stained with Coomassie blue as shown in Fig. 2. When
protein samples ranging from 5 to 25 mg were loaded on SDS-PAGE, only a single band was
detected and such purity was believed to be satis- factory to conduct the further analysis of its glyco-
sylation pattern.
3
.
2
. HPLC separation of glycopeptide According to the deduced amino acid sequence
from the cDNA, cPrx has four potential N-glyco- sylation sites with the consensus sequence of Asn-
X-ThrSer [16]. Studies showed, however, that only three of them Asn-60, Asn-144 and Asn-185
are indeed glycosylated, and the glycans can be separated into three glycopeptides with trypsin
treatment [16]. Therefore, purified cPrx-his6 was digested with TPCK-treated trypsin and Bio-Gel
P-6 filtration chromatography was used to sepa- rate the glycopeptides from the majority of other
tryptic peptides. This eliminated a number of tryp- tic peptide fragments which could have caused
excessive noise on the profiles of the subsequent HPLC separation. As a result, the designation of
glycopeptide peaks on the HPLC profile was sim- plified to a large extent. The elution pattern for
the HPLC separation of glycopeptides from re- combinant cPrx-his6 Fig. 3 is very similar to that
of wild type cPrx on HPLC observed by Sun et al. [19]. A total of three glycopeptides were detected
at similar retention times in the same gradient ranges, and they were also designated as GPa,
GPb and GPc, representing N-185, -60, and -144 [16]. This result indicates that the histidine-tagged
cPrx expressed in transgenic tobacco has the same three glycans as found in wild type cPrx.
3
.
3
. Identification of glycopeptides after HPLC separation
The three tryptic glycopeptides separated on HPLC were further identified by N-terminal se-
quence analysis. A comparison of the amino acid sequencing results, summarized in Table 1, with
Fig. 3. Separation of the three glycopeptides of cPrx-his6 by reverse phase HPLC. The glycopeptides generated by trypsin
digestion and preliminarily purified through Bio-gel filtration were separated by HPLC with a C-18 column as described in
Section 2. The acetonitrile gradient is indicated as dashed lines. Elution was monitored at wavelength 230 nm for pep-
tides. The detected glycopeptides peaks are designated as GPa, GPb and GPc, respectively.
duction of six histidines at its C-terminal had no effect on its catalytic ability. Furthermore, the
cPrx signal peptide was able to direct the secretion of recombinant cPrx into the culture medium as
Table 1 Partial N-terminal sequencing analysis of tryptic glycopeptides obtained by HPLC from cationic peanut peroxidase
a
a
Glycan linkages for GPb and GPc occur at an ASN well beyond the analyzed peptide sequence.
the amino acid sequence deduced from the cPrx suggests strongly that cDNA sequence verified the
occurrence of glycopeptides GPa, GPb and GPc at the glycosylation sites Asn-184, Asn-60 and Asn-
144, respectively. These data provided further evi- dence for the conclusion from earlier experiments
with monoclonal antibodies recognizing the carbo- hydrate-containing motifs [21].
3
.
4
. ABEE deri6atization of sugars The oligosaccharide chains in glycoprotein or
glycopeptides were first hydrolysed by TFA into their monosaccharides. Then the monosaccharides
were coupled to the ultraviolet-absorbing com- pound ABEE by reductive amination with sodium
cyanoborohydride [25]. As a result, each monosac- charide ABEE derivative was detectable at 254 nm
using the UV detector on the HPLC, which gave a good resolution in single chromatographic step.
The derivatization procedure of reducing sugars with ABEE in the presence of sodium cyanoboro-
hydride has some advantages in that it is very simple and results in no side products [25].
3
.
5
. Analysis of monosaccharide composition and construction of standard cur6es for quantitati6e
analysis In N-linked plant glycoproteins some specific
abundant monosaccharides such as Gal, Man, Xyl, Fuc, GlcNAc and GalNAc are present
[14,26 – 28]. But not all these monosaccharides, especially Gal, were always found in plant glyco-
proteins [29,30]. Prior to quantitative analysis, a qualitative
analysis of
monosaccharides was,
therefore, needed to determine the exact compo- nents. Intact protein of cPrx-his6 was hydrolysed
and derivatized, and the sugar derivatives were applied to HPLC using 86 solvent A and 14
Fig. 4. Identification of the monosaccharide components of cPrx-his6 by reverse phase HPLC. Retention times allowed
comparison of the chromatogram of ABEE sugar derivatives from cPrx-his6 hydrolysate with the chromatographic separa-
tion of ABEE derivatives of standard sugars. The identities of the peaks are: 1, GlcNAc; 2, lactose used as an internal
standard for qualitative analysis; 3, Gal; 4, Man; 5, Xyl; 6, Fuc; 7, ABEE reagent. A standard monosaccharide deriva-
tives; B sugar derivatives of cPrx-his6 hydrolysate. Absorp- tion at 254 nm was measured for derivatized sugars.
Fig. 5. Calibration curves for quantitative analysis of monosaccharides. Varying amounts of standard sugar deriva-
tives 200 – 800 nmol in 20-ml aqueous solutions containing 800 nmol lactose were derivatized with ABEE reagent, and
5 of the derivatives was subjected to HPLC. The peak area ratios were expressed relative to the peak area of 40 nmol
lactose.
represent GlcNAc because N-acetylated amino sug- ars like GalNAc and GlcNAc are deacetylated
during the process of hydrolysis, but could be analyzed without reacetylation in single chromato-
graphic step [19,25].
With these results on the monosaccharide of cPrx-his6 as a basis, the quantitative analysis was
started with the construction of standard curves. Aliquots of 20 ml of solutions containing various
amounts 200 – 800 nmol of standard monosaccha- rides and 800 nmol lactose used as an internal
standard were converted to their ABEE derivatives and 5 10 – 40 nmol was subjected to reverse
phase HPLC. Then the average of three determina- tions was used to construct the standard curves. The
high sensitivity and reproducibility of sugar separa- tion provided a linear profile of the standard curves,
when the peak area ratio was expressed, relative to 40 nmol lactose upon reverse phase HPLC separa-
tion of 5 of the sample 10 – 40 nmol Fig. 5.
3
.
6
. Monosaccharide composition of cPrx-his
6
glycans To determine the monosaccharide composition
for individual glycans, the three tryptic glycopep- tides obtained from the trypsin digestion of cPrx
expressed in transgenic tobacco were separated on HPLC, hydrolysed with TFA and ABEE deriva-
tized. Then, the sugar derivatives were subjected to reverse phase HPLC for analysis. Lactose was used
as an internal standard to calculate the peak area ratios of the detected monosaccharides. The molar
percentages calculated according to the standard curves are presented in Table 2. Monosaccharide
components of native cPrx [19], and NMR analysis of the glycan structure [14] show that the native cPrx
glycans are complex glycans. The sugar composi- tion of GPc from cPrx-his6 is essentially the same
as that of native GPc. GPa and GPb in transgenic tobacco have the same amounts of Man, Gal, and
GlcNAc, but they have increased amounts of Fuc and smaller amounts of Xyl. These data strongly
indicate that the complex type of oligosaccharides is present on all the glycosylation sites. Further-
more, a comparison of these data with some of the complex glycan structures established for some
plant glycoproteins [4,30,31] also suggested the existence of complex glycans in cPrx-his6. That is
to say, cPrx in tobacco cells was targeted to ER and proceeded through the processing in the Golgi
compartments.
Table 2 Monosaccharide compositions mol of glycans from cPrx-
his6
a
GlcNAc Glycans
Gal Man
Xyl Fuc
38.4 GPa
12.3 7.7
33.3 8.3
39.8 8.5
GPb 26.9
9.1 15.7
27.9 5.1
12.2 GPc
48.5 6.4
a
Tryptic glycopeptides were separated by HPLC, hy- drolysed and derivatized with ABEE, then the molar percent-
ages were calculated according to the standard curves.
solvent B as a mobile phase in an isocratic mode. Under such conditions, all sugar derivatives were
separated completely and no overlapping peaks were detected. Fig. 4 shows a comparison of the
reverse phase HPLC chromatographic separation of standard sugars, including lactose used as an
internal standard later in the qualitative analysis, with the chromatogram of sugar derivatives from
cPrx-his6. This result shows that five different monosaccharides, GlcNAc, Gal, Man, Xyl and Fuc,
are present in the glycans of cPrx-his6, which is the same number and identity as previously reported for
the native cPrx [19]. For the standard monosaccha- rides, glucosamine GlcN derivative was used to
4. Discussion
