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A rapid sample preparation methodology for plant N-glycan analysis using acid stable PNGase H# Min-Ya Du, Tian Xia, Xiao-Qing Gu, Ting Wang, Hong-Ye Ma, Josef Voglmeir, and Li Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03633 • Publication Date (Web): 08 Nov 2015 Downloaded from http://pubs.acs.org on November 11, 2015
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Journal of Agricultural and Food Chemistry
A rapid sample preparation methodology for plant N-glycan analysis using acid stable PNGase H⁺
Ya M. Dua, Tian Xiaa, Xiao Q. Gua, Ting Wanga, Hong Y. Mab, Josef Voglmeira* and Li Liua*
aGlycomics
and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, China bDepartment
of Plant Pathology, Nanjing Agricultural University, China
*Correspondence
should be addressed to:
E-mail:
[email protected], Fax: +86 25 84399553 Tel: +86 25 84399512 or E-mail:
[email protected]: Fax: +86 25 84399553 Tel: +86 25 84399511 Keywords: N-linked glycosylation, Plant glycan analysis, Core fucosylation; PNGase; Terriglobus; PNGase H⁺
1
Abstact: The quantification of potentially allergenic carbohydrate motifs of plant
2
and insect glycoproteins is increasingly important in biotechnological and
3
agricultural applications due to the use of insect cell-based expression systems and
4
transgenic plants. The need to analyze N-glycan moieties in a highly parallel manner
5
inspired us to develop a quick N-glycan analysis method based on a recently
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discovered bacterial protein N-glycanase (PNGase H⁺). In contrast to the
7
traditionally used PNGase A, which is isolated from almond seeds and only releases
8
N-glycans from proteolytically derived glycopeptides, the herein implemented
9
PNGase H⁺ allows the release of N-glycans directly from the glycoprotein samples.
10
As PNGase H⁺ is highly active under acidic conditions, the consecutive fluorescence
11
labeling step using 2-aminobenzamide (2AB) can be directly performed in the same
12
mixture used for the enzymatic deglycosylation step. All sample handling and
13
incubation steps can be performed in less than four hours and are compatible with
14
microwell-plate sampling, without the need for tedious centrifugation, precipitation
15
or sample transfer steps. The versatility of this methodology was evaluated by
16
analyzing glycoproteins derived from various plant sources using UPLC-analysis,
17
and further demonstrated through the activity analysis of four PNGase H⁺ mutant
18
variants.
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Introduction
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N-glycosylation is a commonly occurring post-translational modification of
21
proteins in eukaryotes.1 Although the initial steps of N-glycan biosynthesis are
22
highly conserved, the decoration of the core carbohydrate structures depends
23
strongly on the glycosidase and glycosyltransferase pool of the individual
24
organism, and show immense diversity in their glycosylation abilities within
25
and between species2-4. Subtle evolutionary alterations in the catalytic ability
26
of these enzymes result in changes in the structural composition of N-glycans.
27
For example, the number, position and linkages of fucose residues in the core
28
region of N-glycans vary significantly between plants, insects and animals.5
29
Although allergic reactions towards pollen, foodstuffs, mites and insect
30
venoms are mainly caused by protein allergens, IgE responses can also be
31
directed towards the carbohydrate portion of insect and plant glyco-
32
proteins.6-7 These carbohydrate motifs, known as cross-reactive carbohydrate
33
determinants (CCDs) obtain their immunogenic potency mainly from
34
structural differences to the endogenously occurring N-glycans in humans.
35
Arguably, the most distinctive modifications of plant and insect N-glycans are
36
the α1,3-linked fucose on the inner core N-acetylglucosamine and/or the
37
β1,2-linked xylose on the innermost core mannose.8 These differences in the
38
posttranslational modification pattern between plants, insects and humans
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are one essential reason why the analysis of N-glycans from plants or insect
40
cell cultures is advisable for therapeutic glycoproteins in the pharmaceutical
41
industry or transgenic crops in agriculture.9 Various strategies have been
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assessed to eliminate or reduce the amount of N-glycans bearing α-1,3 core
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fucose in plants or insect cells.10-11 Finding desired candidate clones requires
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the analysis of large numbers of samples in a short-time and cost-efficient
45
manner.
46
Continuously improving methodologies and the increasing availability of
47
reliable databases led to the rapid development of mammalian N-glycan
48
profiling in recent years.12 The current standard workflow usually consists of
49
protein denaturation, enzymatic N-glycan-release, isolation of N-glycans,
50
fluorescence labeling and subsequent analysis using high performance liquid
51
chromatography (HPLC) and/or mass spectrometry.13 The most widely
52
applied enzyme for releasing N-glycans is recombinant PNGase F (from
53
Flavobacterium meningosepticum; Figure 1, rightmost preparation scheme)
54
which is commercially available and was shown to be efficient towards all
55
types of N-glycans except structures bearing core linked α-1,3 fucose.14
56
Therefore, N-glycans of plants and insects are generally released using
57
PNGase A, which is isolated from almond seeds and shows tolerance towards
58
core modifications of N-glycans. However, PNGase A only releases N-glycans
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from small glycopeptides and therefore requires preliminary treatment of the
60
glycoprotein samples with proteases.15 This step is usually performed in an
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overnight reaction using trypsin or pepsin, resulting in extended sample
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processing times (usually longer than 30 h)16 and increased complexity of the
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N-glycan preparation (Figure 1, preparation scheme in the center).
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Recently we reported the discovery of a novel bacterial protein N-glycanase
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(PNGase H+ from Terriglobus roseus) which also releases core α-1,3 linked
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fucose bearing N-glycans from both glycopeptides and glycoproteins.16 A
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unique feature of this enzyme is that it shows enzymatic activity even in
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concentrated organic acids, which allows the subsequent N-glycan liberation
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and fluorescence labeling steps to be merged into a more rapid sampling
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routine (Figure 1, leftmost preparation scheme). Herein, we present a rapid
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and cost-efficient approach applying recombinant PNGase H+, which only
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requires less than 4 hours sample preparation time.
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Materials and methods
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PNGase treatment
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Recombinant PNGase H+ was expressed and purified as described
77
previously.16 Typically, enzymatic deglycosylation was performed using 10 μL
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assay volumes containing 10 μg of HRP glycoprotein and 10 μUnits of PNGase
79
H+ in 1 M acetic acid. One enzymatic Unit was defined as the activity required
80
to
81
glycopeptide per minute at 37°C in 0.2 M glycine/HCl buffer, pH 2.6. Sample
82
mixtures were incubated for 1 h at 37°C. For quantitative analyses, different
83
amounts of HRP (between 1 μg and 100 μg) were used for deglycosylation
84
experiments. PNGase A released N-glycans were prepared as described
85
previously.16
deglycosylate
1
μmol
of
dabsyl-Gly-Glu-Asn-(GlcNAc4Man3)-Arg
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Glycoprotein isolation from plants
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Plant materials used for the isolation of glycoproteins were purchased at the local
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farmers market: asparagus (Asparagus officinalis), banana (Musa balbisiana), celery
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(Apium graveolens), carrot (Daucus carota), mung bean (Vigna radiata), onion
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(Allium cepa), papaya (Carica papaya), pear (Pyrus bretschneideri), potato (Solanum
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tuberosum) and soya (Glycine max). Approximately 5 g of blended plant foodstuff
92
was centrifuged (20000 g, 20 min, 4°C) to remove insoluble material. Glycoprotein
93
pellets were obtained after precipitation of 1 mL cleared supernatant with 1 mL of
94
aqueous TCA solution (2M) and centrifugation (20000 g, 30 min, 4°C), and directly
95
used for PNGase H+ treatment.
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N-glycan labeling and analysis
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N-glycans were fluorescence labeled using 2-aminobenzamide (2AB) based
98
on the method described by Bigge et al.17 PNGase H+ treated samples were
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mixed with 10 μL of labeling reagent (containing 35 mM of 2-AB and 100 mM
100
NaCNBH3, dissolved in acetic acid/DMSO (30:70, v/v) and incubated for 2 h at
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65°C. Prior to UPLC analysis, 80 μL of acetonitrile was added to the samples.
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2-AB labeled plant N-glycans were separated on a normal-phase UPLC system
103
(Shimadzu Nexera) using an Acquity BEH Glycan column (Waters, 1.7 μm,
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2.1×150 mm) at a flow rate of 0.5 mL/min. The effluent was monitored by
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fluorescence detection (excitation: 330 nm, emission: 420 nm). The column
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temperature was set to 60°C during sample analysis. Ammonium formate in
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water (50 mM, pH 4.5) and 100% acetonitrile were used as solvent A and
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solvent B, respectively. For the analysis of N-glycan standards, a gradient of
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95–78% of B was applied from 0 to 6 min, B was then decreased to 56% over
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39 min followed by further decrease to 0% over 3 min and held at 0% for 2
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min. B was then increased to 95% over 2 min and the column was re-
112
equilibrated to the starting conditions for 8 min (total run time 60 min).
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Standard deviations were determined from three independent experiments.
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N-glycan structures were verified based on their retention time relative to the
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glucose units of an 2AB-labeled dextran standard using the GlycoBase N-
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glycan respiratory.18 UPLC fractions were manually collected between 19.0
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and 20.1 min to isolate the main N-glycan peak observed in the
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chromatogram. After solvent evaporation using vacuum centrifugation,
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samples were subjected to mass spectrometric analysis. Monoisotopic MALDI-
120
TOF-MS spectra were obtained on a Bruker Ultraflex Extreme TOF-TOF
121
spectrometer with 6-aza-2-thiothymine as matrix. Mass spectra were
122
processed using Flexanalysis version 3.3. MS data were analyzed manually,
123
and the mass peaks from MS and MS-MS spectra were further evaluated on
124
GlycoWorkbench version 1.1.19
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Stability of plant N-glycans in acetic acid
126
2-AB labeled N-glycans (10 μL, derived from 20 μg of HRP as described above)
127
were incubated in 200 μL of 1 M acetic acid for 2 h, 4 h, 8 h and 12 h. 20 μL
128
were taken out and mixed with 80 μL of acetonitrile prior to UPLC injection.
129
Labeled HRP-derived N-glycans without incubation in acidic conditions were
130
used as controls. The stability of labeled glycans was calculated based on the
131
peak areas of the main N-glycan structure (MMXF3).
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Generation of PNGase H+ point mutations
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PNGase H+ point mutations were generated according to the QuickChange XL
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Site-Directed Mutagenesis protocol (Stratagene) using the primers listed in
136
Table S1 in the supporting information. The mutated plasmids were verified
137
by DNA sequencing and transformed into E. coli BL21(DE3) competent cells
138
(Invitrogen) for recombinant expression. The transformants (including a non-
139
mutated wild-type PNGase H+ containing transformant) were cultured at 37°C
140
in 400 mL LB media at 250 rpm. After the culture density reached an OD600
141
value of 0.8, IPTG was added to a final concentration of 0.5 mM and bacterial
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cultures were grown at 18°C overnight. Cells were collected by centrifugation
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at 5000 g for 10 min and resuspended in 5 mL of cell lysis buffer (50 mM
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Tris/HCl (pH 7.5), 50 mM NaCl, 1% Triton X-100 (v/v) and 1 mM PMSF). Cell
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lysis was performed using ultrasonication at 4°C (40 on/off cycles with 20 μm
146
amplitude and 15 s intervals) and the cell debris were then separated by
147
centrifugation (20000 g, 20 min, 4°C). Supernatants from each mutant (2 μL)
148
were tested using the methods described above using HRP as substrate.
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150
Results and discussion
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Enzymatic glycan release and 2AB-labeling
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General protocols for N-glycan release require reaction buffers for the
153
enzymatic deglycosylation reaction, resulting in high amounts of residual
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buffer salts after sample concentration. As high amounts of salts were claimed
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to hinder the subsequent N-glycan fluorescence labeling using reductive
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amination, a desalting step for binding/eluting the N-glycans using porous
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graphitized carbon (PGC) has to be included into the sample workup.20 To
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minimize the amount of buffer salts in the deglycosylation mixture, the
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previously used glycine-HCl buffer was replaced with acetic acid at various
160
concentrations.
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Six different final concentrations between 50 mM and 5 M acetic acid were
162
tested. As shown in Figure 2A, the results revealed that 1 M acetic acid gave
163
the best results for the enzymatic glycan release and labeling efficiency. In
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comparison, the enzymatic release and labeling reactions performed in 50
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mM, 100 mM and 500 mM, 2 M and 5 M acetic acid showed relative labeling
166
efficiencies of 5%, 14%, 81%, 76% and 11%, respectively. The glycine-HCl
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buffer used for enzymatic deglycosylation reactions in previous experiments
168
only showed 2% of overall N-glycan deglycosylation/labeling efficiency in
169
comparison to the assay performed in 1 M acetic acid. Therefore, we decided
170
to use 1 M acetic acid for the following digestion/labeling experiments.
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Typically, enzymatic incubation times for the release of N-glycans using
172
commercial preparations of PNGase F require between 2 h and 16 h, and
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commercial PNGase A incubation times of 16 h are recommended. Longer
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incubation times might be chosen to ensure quantitative N-glycan release or
175
for the convenience of the analysts’ workflow, as 16 h reactions may be
176
performed overnight. We tried to further reduce the enzymatic N-glycan
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digestion times. Figure 2B shows that for the digestion of 10 μg denatured
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HRP with 5 μUnits of recombinant PNGase H+, the amount of released N-
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glycan increased rapidly during the first hour. As N-glycan release only
180
increased slightly between 2 h and 8 h incubation time, we concluded that a 2
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h incubation time should be sufficient for the consecutive 2AB-labeling
182
reaction. Furthermore, monitoring the deglycosylation of HRP in the
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optimized release condition by SDS-PAGE demonstrated that the majority of
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N-glycans can be released within 2 h incubation time (Supplementary Figure
185
S1).
186 187
N-glycan analysis
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Core α-1,3 fucosylation of N-glycans is a common epitope of plant and insect
189
glycoproteins,
190
pharmaceutical products and foodstuffs from these origins is of high interest.
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Horseradish Peroxidase (HRP) was selected as a model glycoprotein. The N-
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glycan profile of this glycoprotein is well studied, with most of its nine N-
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glycosylation sites bearing core α-1,3 fucose moieties.21 Approximately 70-
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80%
195
modifications.22 As shown in Figure 3A, the obtained UPLC profile of the
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released N-glycans using PNGase H+ are in good agreement with expected N-
197
glycan profile of HRP using PNGase A. Among various smaller peaks, one
198
dominant peak eluting at 19.8 min contributed approximately 80% of the
199
total fluorescence signal. This peak fraction was manually collected and
of
the
and
total
so
the
HRP
qualitative
N-glycan
and
pool
quantitative
are
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of
(Xyl)Man3(Fuc)GlcNAc2
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further analyzed by MALDI-TOF mass spectrometry. The m/z value of
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1331.446 Da compares well with the theoretical monoisotopic mass of 2AB-
202
Hex3HexNAc2Xyl1Fuc1 ([M+Na]+: 1331.48, Figure 3B). MS-MS fragmentation
203
further confirmed its N-glycan composition, showing that fucose was attached
204
to the proximal GlcNAc of the chitobiose core and xylose to the core
205
trimannoside (data not shown). Both UPLC chromatograms and mass
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spectrometric analysis verified that this quick preparation of HRP with
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recombinant PNGase H+ is suitable for glycoprotein samples containing core
208
α-1,3 fucose.
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The N-glycan profiles of ten selected plant glycoproteins prepared with this
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rapid labeling methodology is in good agreement with reported N-
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glycoprofiles using conventional PNGase A treatment23, with the dominant
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glycan portions bearing core xylose and/or fucose (Figure 4 and Table S2).
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Stability of plant N-glycans in acetic acid
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Previous studies showed that some sugar moieties in N-glycans such as sialic
215
acids are labile under acidic conditions. Therefore, fluorescence labeling with
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2AB-catalyzed by acetic acid may also lead to partial de-glycosylation of N-
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glycans.24 Although both steps, the proteolytic sample treatment with pepsin
218
and the N-glycan release using PNGase A, are usually performed in acidic
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buffers (pH 4-5), Takahashi et al reported that no hydrolysis of sialic acid was
220
observed under these conditions.25 However, as recombinant PNGase H+
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shows highest activity at pH 2.6, we wanted to determine whether the acidic
222
conditions used for our method affect the stability of the released plant N-
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glycans. Therefore, 2AB-labeled samples were incubated in 1 M acidic acid for
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different time intervals (0 h, 2 h, 4 h, 8 h and 12 h). Comparison of the
225
different N-glycan profiles showed that the area of the largest peak (MMXF3,
226
retention time = 19.8 min) showed no significant decrease over time (Figure
227
5A). This indicates that N-glycan release in 1 M acetic acid had little effect on
228
the stability of 2AB-labeled N-glycans derived from plants.
229 230
Quantitative N-glycan analysis
231
After optimization and verification of the rapid N-glycan analysis method, a
232
calibration curve for different amounts of HRP within the range of 1 μg and 20
233
μg demonstrated high linearity (R2=0.9964), clearly confirming the utility and
234
consistency of the method (Figure 5B). However, peak areas of tested HRP
235
standards above 20 μg glycoprotein (50 μg and 100 μg, respectively) did not
236
show the same linear behavior, and therefore the work range for glycoprotein
237
analysis should be set below 20 μg of glycoprotein. The obtained data
238
furthermore suggested that 1 μg of HRP was already sufficient for the
239
quantitative N-glycan analysis using our procedure.
240
241
Rapid screen of recombinant PNGase H+ mutant variants
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This quick preparation method could be applied not only to the analysis of N-
243
linked carbohydrates from plants, but also to the rapid screening of enzyme
244
candidates with PNGase H+-like activity, or studying point mutations of
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recombinant PNGase H+ variants. Therefore, we decided to perform a
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mutational study on glutamic acid residues which are potentially involved in
247
the acid/base assisted deglycosylation mechanism of the enzyme.26 Based on
248
the conserved amino acid sequence of PNGase H+, we designed four different
249
point mutants changing different glutamic acids to alanines (Glu126Ala,
250
Glu239Ala, Glu263Ala and Glu293Ala, Supplementary Figure S2) using site-
251
directed mutagenesis. The wild-type and mutant variant enzymes were
252
directly obtained for analysis from crude E. coli cell lysate without further
253
purification. Figure 5C shows that both mutants Glu239Ala and Glu263Ala
254
had no apparent activity, whereas Glu126Ala and Glu293Ala showed
255
comparable activities to wild-type PNGase H+. These results indicate the
256
functional importance of the residues Glu239 and Glu263, and the
257
applicability of this rapid N-glycan analysis method to activity screens.
258 259
Conclusive Remarks
260
We have developed a novel and rapid preparation method for N-glycans
261
based on recombinant PNGase H+, which was suitable for the production of N-
262
glycan profiles of glycoproteins from various plant materials. The full
263
procedure could be finished in less than 4 h and all sampling steps are
264
compatible with automatic handling in microwell plates, without the need for
265
centrifugation, precipitation or sample-transfer steps. We verified this
266
methodology using HRP as a model glycoprotein, and quantified the major
267
HRP N-glycan MMXF3. Analyzing various amounts of HRP showed high
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linearity in a range from 1 μg to 20 μg, which clearly confirms the robustness
269
and utility of this method. The application of this rapid sampling method to
270
the analysis of N-glycans from other biological sources such as milk or blood
271
serum is one of our future research targets.
272 273
Abbreviations
274
2AB, 2-aminobenzamide; CCDs, cross-reactive carbohydrate determinants;
275
DMSO, dimethyl sulfoxide; Fuc, fucose; GlcNAc, N-acetyl-D-glucosamine; HCl,
276
hydrochloric acid; HRP, horse radish peroxidase; IgE, Immunoglobulin E;
277
IPTG, isopropyl-β-D-thiogalactopyranoside; MALDI, matrix-assisted laser
278
desorption/ionization; Man, mannose; MS, mass spectrometry; m/z, mass per
279
charge; PMSF, phenyl-methanesulfonyl fluoride; TCA, trichloroacetic acid;
280
TOF, time of flight; Tris, tris(hydroxymethyl)aminomethane; UPLC, ultra-
281
performance liquid chromatography; Xyl, xylose.
282 283
Acknowledgements
284
The authors would like to thank Prof. Yuanchao Wang for access to the Bruker
285
Ultraflex MALDI-TOF Mass Spectrometer and Dr. Louis Conway (GGBRC,
286
Nanjing) for language editing of this manuscript.
287 288
Funding Sources
289
This work was supported in parts by the Natural Science Foundation of China
290
(grant number 31471703 to L.L. and J.V., A0201300537 to J.V. and L.L., and
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BK20140719 to L.L. and T.W.), by the Jiangsu Provincial Natural Science
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Foundation of China (Project: BK20140719 to W.T. and L.L.) and the 100
293
Foreign Talents Plan (grant number JSB2014012 to J.V.).
294 295
Supporting Information Available: Primers and protein sequences for
296
mutational studies. This material is available free of charge via the Internet at
297
http://pubs.acs.org
298 299
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Figure Captions
Figure 1: Overview of N-glycan preparation schemes for various biological materials using different PNGases.
Figure 2: Labeling efficiency of HRP N-glycans using (A) glycine-HCl-buffer/acetic acid or (B) different time intervals.
Figure 3: Released N-glycans from HRP (A). The UPLC chromatograms of released N-glycans using PNGase H+ (top panel) and PNGase A (bottom panel. The arrow indicates the main N-glycan portion MMXF3. The glycan peak labeled with an asterisk is presumably a side product of the PNGase A deglycosylation reaction. (B) MALDI-TOF-MS analysis of the collected MMXF3 fraction.
Figure 4: Evaluation of the N-glycan method. (A) Stability of plant N-glycans in 1 M acetic acid after various time intervals. (B) Calibration curve of HRP derived Nglycans after 2AB-labeling. (C) Rapid screen of recombinant PNGase H+ wild-type and mutant variants.
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