Rapid Sample Preparation Methodology for Plant N-Glycan Analysis

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Article

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

ACS Paragon Plus Environment


6

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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19

Introduction

20

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

39

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



42

assessed to eliminate or reduce the amount of N-glycans bearing α-1,3 core

43

fucose in plants or insect cells.10-11 Finding desired candidate clones requires

44

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

59

from small glycopeptides and therefore requires preliminary treatment of the

60

glycoprotein samples with proteases.15 This step is usually performed in an

61

overnight reaction using trypsin or pepsin, resulting in extended sample

62

processing times (usually longer than 30 h)16 and increased complexity of the

63

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

65

(PNGase H+ from Terriglobus roseus) which also releases core α-1,3 linked

66

fucose bearing N-glycans from both glycopeptides and glycoproteins.16 A

67

unique feature of this enzyme is that it shows enzymatic activity even in

68

concentrated organic acids, which allows the subsequent N-glycan liberation

69

and fluorescence labeling steps to be merged into a more rapid sampling

70

routine (Figure 1, leftmost preparation scheme). Herein, we present a rapid

71

and cost-efficient approach applying recombinant PNGase H+, which only

72

requires less than 4 hours sample preparation time.

73

74

Materials and methods

75

PNGase treatment

76

Recombinant PNGase H+ was expressed and purified as described

77

previously.16 Typically, enzymatic deglycosylation was performed using 10 μL

78

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



86

Glycoprotein isolation from plants

87

Plant materials used for the isolation of glycoproteins were purchased at the local

88

farmers market: asparagus (Asparagus officinalis), banana (Musa balbisiana), celery

89

(Apium graveolens), carrot (Daucus carota), mung bean (Vigna radiata), onion

90

(Allium cepa), papaya (Carica papaya), pear (Pyrus bretschneideri), potato (Solanum

91

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.

96

N-glycan labeling and analysis

97

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

99

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

101

65°C. Prior to UPLC analysis, 80 μL of acetonitrile was added to the samples.

102

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,

104

2.1×150 mm) at a flow rate of 0.5 mL/min. The effluent was monitored by

105

fluorescence detection (excitation: 330 nm, emission: 420 nm). The column

106

temperature was set to 60°C during sample analysis. Ammonium formate in

107

water (50 mM, pH 4.5) and 100% acetonitrile were used as solvent A and

108

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

110

39 min followed by further decrease to 0% over 3 min and held at 0% for 2

111

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).

113

Standard deviations were determined from three independent experiments.

114

N-glycan structures were verified based on their retention time relative to the

115

glucose units of an 2AB-labeled dextran standard using the GlycoBase N-

116

glycan respiratory.18 UPLC fractions were manually collected between 19.0

117

and 20.1 min to isolate the main N-glycan peak observed in the

118

chromatogram. After solvent evaporation using vacuum centrifugation,

119

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

125

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).



132 133

Generation of PNGase H+ point mutations

134

PNGase H+ point mutations were generated according to the QuickChange XL

135

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

142

cultures were grown at 18°C overnight. Cells were collected by centrifugation

143

at 5000 g for 10 min and resuspended in 5 mL of cell lysis buffer (50 mM

144

Tris/HCl (pH 7.5), 50 mM NaCl, 1% Triton X-100 (v/v) and 1 mM PMSF). Cell

145

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.

149

150

Results and discussion

151

Enzymatic glycan release and 2AB-labeling

152

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

155

to hinder the subsequent N-glycan fluorescence labeling using reductive

156

amination, a desalting step for binding/eluting the N-glycans using porous

157

graphitized carbon (PGC) has to be included into the sample workup.20 To

158

minimize the amount of buffer salts in the deglycosylation mixture, the

159

previously used glycine-HCl buffer was replaced with acetic acid at various

160

concentrations.

161

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

164

comparison, the enzymatic release and labeling reactions performed in 50

165

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

167

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.

171

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

173

commercial PNGase A incubation times of 16 h are recommended. Longer

174

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

178

HRP with 5 μUnits of recombinant PNGase H+, the amount of released N-

179

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

181

h incubation time should be sufficient for the consecutive 2AB-labeling

182

reaction. Furthermore, monitoring the deglycosylation of HRP in the

183

optimized release condition by SDS-PAGE demonstrated that the majority of

184

N-glycans can be released within 2 h incubation time (Supplementary Figure

185

S1).

186 187

N-glycan analysis

188

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.

191

Horseradish Peroxidase (HRP) was selected as a model glycoprotein. The N-

192

glycan profile of this glycoprotein is well studied, with most of its nine N-

193

glycosylation sites bearing core α-1,3 fucose moieties.21 Approximately 70-

194

80%

195

modifications.22 As shown in Figure 3A, the obtained UPLC profile of the

196

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


analysis

of

(Xyl)Man3(Fuc)GlcNAc2

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200

further analyzed by MALDI-TOF mass spectrometry. The m/z value of

201

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

206

spectrometric analysis verified that this quick preparation of HRP with

207

recombinant PNGase H+ is suitable for glycoprotein samples containing core

208

α-1,3 fucose.

209

The N-glycan profiles of ten selected plant glycoproteins prepared with this

210

rapid labeling methodology is in good agreement with reported N-

211

glycoprofiles using conventional PNGase A treatment23, with the dominant

212

glycan portions bearing core xylose and/or fucose (Figure 4 and Table S2).

213

Stability of plant N-glycans in acetic acid

214

Previous studies showed that some sugar moieties in N-glycans such as sialic

215

acids are labile under acidic conditions. Therefore, fluorescence labeling with

216

2AB-catalyzed by acetic acid may also lead to partial de-glycosylation of N-

217

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

219

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+

221

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-



223

glycans. Therefore, 2AB-labeled samples were incubated in 1 M acidic acid for

224

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

242

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

246

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



268

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

292

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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Figures

Figure 1 (two columns)



Figure 2 (one column)


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Figure 4 (two columns)


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Graphic for table of contents


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Rapid Sample Preparation Methodology for Plant N-Glycan Analysis

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