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Nov 8, 2015 - Plant Pathology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People,s Republic of China. •S Supporting Information. ABST...
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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

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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

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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

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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

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cell cultures is advisable for therapeutic glycoproteins in the pharmaceutical

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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

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manner.

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Continuously improving methodologies and the increasing availability of

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reliable databases led to the rapid development of mammalian N-glycan

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profiling in recent years.12 The current standard workflow usually consists of

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protein denaturation, enzymatic N-glycan-release, isolation of N-glycans,

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fluorescence labeling and subsequent analysis using high performance liquid

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chromatography (HPLC) and/or mass spectrometry.13 The most widely

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applied enzyme for releasing N-glycans is recombinant PNGase F (from

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Flavobacterium meningosepticum; Figure 1, rightmost preparation scheme)

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which is commercially available and was shown to be efficient towards all

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types of N-glycans except structures bearing core linked α-1,3 fucose.14

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Therefore, N-glycans of plants and insects are generally released using

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PNGase A, which is isolated from almond seeds and shows tolerance towards

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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

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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

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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

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H+ in 1 M acetic acid. One enzymatic Unit was defined as the activity required

80

to

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glycopeptide per minute at 37°C in 0.2 M glycine/HCl buffer, pH 2.6. Sample

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mixtures were incubated for 1 h at 37°C. For quantitative analyses, different

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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

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was centrifuged (20000 g, 20 min, 4°C) to remove insoluble material. Glycoprotein

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pellets were obtained after precipitation of 1 mL cleared supernatant with 1 mL of

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aqueous TCA solution (2M) and centrifugation (20000 g, 30 min, 4°C), and directly

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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

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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

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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

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(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-

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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-

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TOF-MS spectra were obtained on a Bruker Ultraflex Extreme TOF-TOF

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spectrometer with 6-aza-2-thiothymine as matrix. Mass spectra were

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processed using Flexanalysis version 3.3. MS data were analyzed manually,

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and the mass peaks from MS and MS-MS spectra were further evaluated on

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GlycoWorkbench version 1.1.19

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Stability of plant N-glycans in acetic acid

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2-AB labeled N-glycans (10 μL, derived from 20 μg of HRP as described above)

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were incubated in 200 μL of 1 M acetic acid for 2 h, 4 h, 8 h and 12 h. 20 μL

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were taken out and mixed with 80 μL of acetonitrile prior to UPLC injection.

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Labeled HRP-derived N-glycans without incubation in acidic conditions were

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used as controls. The stability of labeled glycans was calculated based on the

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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

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Table S1 in the supporting information. The mutated plasmids were verified

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by DNA sequencing and transformed into E. coli BL21(DE3) competent cells

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(Invitrogen) for recombinant expression. The transformants (including a non-

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mutated wild-type PNGase H+ containing transformant) were cultured at 37°C

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in 400 mL LB media at 250 rpm. After the culture density reached an OD600

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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

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amplitude and 15 s intervals) and the cell debris were then separated by

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centrifugation (20000 g, 20 min, 4°C). Supernatants from each mutant (2 μL)

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were tested using the methods described above using HRP as substrate.

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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

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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

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concentrations.

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Six different final concentrations between 50 mM and 5 M acetic acid were

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tested. As shown in Figure 2A, the results revealed that 1 M acetic acid gave

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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

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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

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only showed 2% of overall N-glycan deglycosylation/labeling efficiency in

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comparison to the assay performed in 1 M acetic acid. Therefore, we decided

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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

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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

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for the convenience of the analysts’ workflow, as 16 h reactions may be

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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

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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

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glycoproteins,

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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%

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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-

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glycan profile of HRP using PNGase A. Among various smaller peaks, one

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dominant peak eluting at 19.8 min contributed approximately 80% of the

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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

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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

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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

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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

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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

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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

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different N-glycan profiles showed that the area of the largest peak (MMXF3,

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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

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After optimization and verification of the rapid N-glycan analysis method, a

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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

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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

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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

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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

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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

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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

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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,

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Nanjing) for language editing of this manuscript.

287 288

Funding Sources

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This work was supported in parts by the Natural Science Foundation of China

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(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.).

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Supporting Information Available: Primers and protein sequences for

296

mutational studies. This material is available free of charge via the Internet at

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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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