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Functional Structure/Activity Relationships

Ultrasonic pretreatment combined with dry-state glycation reduced the IgE/IgG-binding ability of #lactalbumin revealed by high-resolution mass spectrometry Jun Liu, Zong-cai Tu, Guang-xian Liu, Chen-di Niu, Hong-lin Yao, Hui Wang, Xiao-mei Sha, Yan-hong Shao, and Igor A. Kaltashov J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00489 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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Page 1 of 34

Journal of Agricultural and Food Chemistry

1

Ultrasonic pretreatment combined with dry-state glycation reduced

2

the IgE/IgG-binding ability of α-lactalbumin revealed by

3

high-resolution mass spectrometry

4 5

Jun Liua, Zong-cai Tua,b*, Guang-xian Liua, Chen-di Niuc, Hong-lin Yaoc, Hui Wangb*,

6

Xiao-mei Shaa, Yan-hong Shaoa, Igor A. Kaltashovc

7 8 9

a

College of Life Sciences, Jiangxi Normal University, Nanchang, Jiangxi 330022,

10

China;

11

b

12

Nanchang, Jiangxi 330047, China;

13

c

14

01003, USA

15

*

16

E-mail:

17

(Hui-Wang)

State Key Laboratory of Food Science and Technology, Nanchang University,

Department of Chemistry, University of Massachusetts-Amherst, Amherst, MA,

Corresponding authors. Tel.: +86-791-8812-1868; fax: +86-791-8830-5938. [email protected]

(Zong-cai

Tu),

18 19 20 21 22 23

ACS Paragon Plus Environment

[email protected]


24

Abstract: Bovine α-lactalbumin (α-LA) is one of major food allergens in cow's

25

milk. The present work sought to research the effects of ultrasonic pretreatment

26

combined with dry heating-induced glycation between α-LA and galactose on the

27

IgE/IgG-binding ability and glycation extent of α-LA, which determined by inhibition

28

ELISA and high-resolution mass spectrometry respectively. The IgE/IgG-binding

29

ability of glycated α-LA were significantly decreased as a result of ultrasonic

30

pretreatment, while average molecular weight, incorporation ratio (IR) value, the

31

location and number of glycation site, and degree of substitution per peptide (DSP)

32

value was elevated. When the mixtures of α-LA and galactose pretreated by

33

ultrasonication at 150 W/cm2, the glycated α-LA possess seven glycation sites, the

34

highest IR and DSP value, and the lowest IgE/IgG-binding ability. Therefore, the

35

decrease in IgE/IgG-binding ability of α-LA depend not only on the shielding effect

36

of the linear epitope was found to be caused by the glycation of K13, K16, K58, K93

37

and K98 sites, but also on the intensified glycation extent, which reflected in the

38

increase of IR value, the number of glycation sites and DSP value. Moreover,

39

allergenic proteins and monosaccharides pretreated by ultrasonication then followed

40

by dry-state glycation was revealed as a promising way of achieving lower

41

allergenicity of proteins in food processing.

42 43

Keywords: Alpha-lactalbumin, IgE/IgG-binding ability, ultrasonication, glycation, mass

44

spectrometry


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45

Introduction

46

Bovine α-lactalbumin (α-LA) is a potential allergen that causes about 30-35%

47

IgE-mediated cow's milk allergy1. Native α-LA is a 14.2 kDa Ca-binding protein,

48

which has 123 amino acid residues (AA) 2. It consists of a α-helical domain and a

49

β-sheet domain, which are connected by a calcium binding loop3. α-LA strongly binds

50

metal ions, possess a variety of such useful functional characteristics as

51

immune-modulating, antioxidant, antibacterial or antitumor activity4-6. The antigenic

52

site of α-LA was located on sequence 5-182, and a high IgE-binding ability is

53

associated with sequence (AA 17-58) of bovine α-LA7. Reported methods, such as

54

heat

55

non-enzymatic glycosylation (glycation)10, 11 rationally decreased the allergenicity of

56

α-LA. For these processes, glycation is the early stage of Maillard reaction, and

57

mainly occurs between a carbonyl group of saccharides and an amino group (Lys and

58

Arg) of proteins. It can improve the structural and physicochemical properties of

59

proteins11-14, for example, antioxidant ability, emulsifying, and foaming property,

60

especially for decreasing the allergenicity of α-LA14,

61

supplement for infant formulae which undergo the modification of glycation, the

62

functionality of α-LA was modulated in food processing. However, a single glycation

63

cannot reduce the allergenicity of α-LA to a satisfactory result.

treatment8,

gamma

irradiation1,

high-intensity

ultrasonication9

and

15

. α-LA can be used as a

64

Ultrasonication, a non-thermal processing technology, which can be used to

65

improve the glycation reaction16 and develop a new product with an unique

66

functionality17 in four characteristic ways18, including heating effects, acoustic



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67

cavitation,

68

ultrasonication can disrupt the structure of α-LA19, 20, and glycation was strongly

69

decreased the IgE/IgG-binding ability of α-LA21, no study on the effects of ultrasonic

70

pretreatment coupled with glycation reaction on the IgE/IgG-binding ability of α-LA.

acoustic

streaming,

and

fluid

particles

oscillations.

Although

71

The shielding effects of the linear epitopes was caused by glycation reaction

72

between allergenic proteins and sugar, leading to influence the IgE/IgG-binding

73

ability of allergenic proteins. Glycation occurs on the Lys, Arg and N-terminal amino

74

acid of proteins and alters the peptides. The major α-LA epitopes probably contain

75

one or more Lys and Arg residues. Thus, the location of glycation site was associated

76

with the IgE/IgG-binding ability of α-LA. But, whether the change of the

77

IgE/IgG-binding ability of α-LA treated by ultrasonic pretreatment combined with

78

glycation are due to location and number of glycation sites is still uncertain. Moreover,

79

studies on the relationship between the IgE/IgG-binding ability and glycation extent

80

of α-LA that was ultrasonicated by ultrasonication and subjected to glycation are rare.

81

At present, high-resolution mass spectrometry such as Q Exactive mass spectrometer

82

can be used to investigate their relationships due to it can exactly analyse the number

83

and location of glycation site and glycation extent per site in the protein.

84

In the present study, ultrasonication was used to pretreat α-LA, aiming to study

85

the impact of ultrasonication on the IgE/IgG-binding ability, and on structural

86

properties of glycated α-LA. The first, the IgE/IgG-binding ability of glycated α-LA

87

was detected by inhibition ELISA. The glycation extent (illustrated by average

88

molecular weight, incorporation ratio value, the location and number of glycation


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89

sites, and degree of substitution per peptide value) of glycated α-LA were determined

90

using high-resolution mass spectrometry. Our study results understood that glycation

91

extent plays an essential role in reducing the IgE/IgG-binding ability of α-LA treated

92

by ultrasonic pretreatment coupled with glycation, thus provide basic information on

93

the potential applications of α-LA in the food industry and dairy products.

94

Materials and methods

95

Chemicals and materials

96

Alpha-lactalbumin (α-LA) from bovine milk (L5385, Type I, ≥ 85%), galactose

97

(G0625), pepsin (P6887, 3,200-4,500 units/mg protein), and Goat anti-human

98

IgE-HRP conjugate (A9667), mass Standards Kit for the 4700 Proteomics Analyzer

99

(AB SCIEX, 4333604) were from Sigma-Aldrich (St. Louis, MO). Goat anti-rabbit

100

IgG-HRP conjugate (SE131) was from Beijing Solarbio Technology Co., Ltd (Beijing,

101

China).

102

Ten sera from patients allergic to milk were from Plasma Lab International

103

(Everett, USA). They had total milk protein-specific IgE levels ranging from 5.74 to

104

78.6 KUA/L. Human antisera (prepared by mixed ten patient’s sera at same volume)

105

was applied to study the IgE-binding abilities of α-lactalbumin. Rabbit antisera was

106

prepared using a previously reported protocol1.

107

Sample Preparation

108

1.0 mg/mL of α-LA solution was prepared by dissolving native α-LA in 50 mM



109

phosphate buffer saline (PBS), and the pH of this solution was set to 7.4. Native α-LA

110

was used as the control. 10 mL of α-LA solution were transferred into 25 mL flat

111

bottom conical flasks which were immersed in the ice bath, followed by

112

ultrasonicated using a Q700 Sonicator (microtip probe (1/8 in. = 3 mm) with a 9s on

113

and 1s off pulsation at an actual ultrasonic intensity of 0, 90 and 150 W/cm2 for 15

114

min, respectively. Then, 10 mg of galactose (Gal) was prepared in 10 mL of native

115

and treated α-LA solution, respectively. 10 mg of galactose were dispersed in 10 mL

116

of native α-LA solution, and the subsequent procedure was the same to ultrasonic

117

treatment. Native α-LA solution, native α-LA -Gal solution, ultrasonicated α-LA -Gal

118

solution and ultrasonicated (α-LA -Gal) solution were freeze-dried into the powders,

119

followed by incubation at 55 oC and 79% relative humidity (saturated potassium

120

chloride solution) for 4 h. The reaction was stopped in an ice bath, then the samples

121

was filtered using a Centricon (Millipore) centrifugal filters with 3000 Da to remove

122

unreacted galactose and salts. The concentration of all the samples were diluted into

123

5.0 mg/mL for future use. Native α-LA was named N-LA. α-LA was treated by

124

ultrasonication at 0, 90 and 150 W/cm2 then glycation was named N-LA-G,

125

U-LA-G-90, U-LA-G-150, respectively. The mixtures of α-LA and Gal were treated

126

by ultrasonication at 90 and 150 W/cm2 then glycation was named U-(LA-G)-90 and

127

U-(LA-G)-150. The treatments were performed in triplicates. Schematic depiction of

128

the sample preparation was shown in Fig. 1.

129

IgE/IgG-binding ability determination


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Inhibition ELISA assays were used to estimate the IgE/IgG-binding ability of α-LA

131

by the method of Chen et al.22, with human antisera and rabbit antisera, respectively.

132

The 96-wells microtitre plates were coated with 4 µg/mL of native α-LA samples

133

(100 µL/well) and followed by incubation at 4 oC for overnight. The plates were

134

washed three times by addition of PBST (prepared by dissolving 0.05% Tween-20 in

135

10 mM PBS) and followed by blocking by addition of 3% mg/mL fish gelatin

136

(dissolved in carbonate buffer) for 1 h at 37 oC, then again washed. The incubation at

137

37 oC for 2 h was initiated by addition of 50 µL of antisera samples (1:10 diluted

138

human sera or 1:10 000 diluted rabbit sera) and 50 µL of the treated samples. After

139

incubation, removed the solution, washed the plate. 100 µL of purified goat

140

anti-human IgE-HRP conjugate or goat anti-rabbit IgG-HRP conjugate (diluted into

141

1:5000 in PBST) were added, then incubated at 37 oC for 1 h. After incubation,

142

tetramethylenbenzidine solution (100 µL) were immediately added to each well, and

143

the reaction was stopped by addition of sulfuric acid (50 µL, 2 mol/L). Finally, the

144

absorption was monitored at 450 nm by a microplate reader (BioTek Instruments Co.

145

Ltd., Vermont). Decline rate was calculated: % inhibition = 1 − × 100 ,

146

where B and B0 are the absorbance value of the well with and without glycation

147

samples, separately. Each sample was performed in triplicates.

148

MALDI-TOF analysis

149

Molecular weight (MW) of all the glycated samples were analyzed using

150

MALDI-TOF mass spectrometer (AB Science, USA) according to a previously



Page 8 of 34

151

reported method16. Matrix was prepared by dissolving 5.0 mg/mL of sinapic acid in

152

50% acetonitrile with 0.1% trifluoroacetic acid. The proteins were diluted into 1:100

153

with ultrapure water. 1.5 µL of mixtures at 1:1 protein solution to matrix ratio was

154

spotted onto MALDI target, then flowed by air-drying. Calibration Mixture 1 was set

155

as a standard calibration, error is less than 0.5 Da. The spectrum was acquired in

156

5000-50000 m/z range with 500 laser shots were accumulated for each measurement.

157

Data Explorer (TM) Software was applied to analyze the mass spectrometric data.

158 159

In this work we used incorporation ratio (IR) to analyze the degree of protein glycation. IR of Gal to α-LA can be estimated using the following equation:

160

IR =

MW

− MW"#$ 162.0528 !

!

161

where 162.0528 is molecular weight of Gal attached to α-LA, MWGlycated α-LA is the

162

MW of glycated α-LA, and MWUnglycated α-LA is the molecular weight of unglycated

163

α-LA.

164

Identification of glycation sites

165

The glycation sites of glycated α-LA were identified by our previous method23.

166

Protein digestion was prepared by filter-aided sample preparation method. After

167

filter-aided sample preparation, the peptides were separated with an Agilent 1200

168

HPLC (Agilent Technologies, USA) using a C18 column, then the column effluent

169

was performed by ETD-MS/MS for samples analysis, as previously reported setup

170

and method23.


Page 9 of 34


171 172

We applied the degree of substitution per peptide of each site (DSP) to analyze glycation extent. DSP can be estimated using the following equation24: ∑#/34 i × I../0 / × 12 DSP = ∑#/34 I../0 / × 12

173

where I is the sum of the intensity of glycated peptide, and i is the number of

174

galactose units attached to the peptide in each glycated form.

175

Results and Discussion

176

Analysis of IgE/IgG-binding ability

177

The IgE/IgG-binding ability of N-LA, N-LA-G, U-(LA-G)-90, U-(LA-G)-150,

178

U-LA-G-90 and U-LA-G-150 were determined with inhibition ELISA assays. The

179

IgE/IgG-binding ability was reflected by IC50 value. The results are listed in Fig. 2.

180

The IC50 value of U-(LA-G)-90, U-(LA-G)-150, U-LA-G-90 and U-LA-G-150 shifted

181

to 14.5, 16.2, 12.2, and 14.2 µg/mL, respectively, much higher than the IgE-binding

182

ability of N-LA and N-LA-G, which were 4.5 and 11.1 µg/mL (Fig. 2A). Interestingly,

183

Fig. 2B shows a similar trend. The IC50 value of N-LA-G (3.9 µg/mL), U-(LA-G)-90

184

(5.5 µg/mL), U-(LA-G)-150 (6.4 µg/mL), U-LA-G-90 (4.6 µg/mL) and U-LA-G-150

185

(5.1 µg/mL) were higher than that of N-LA (1.1 µg/mL). These results indicated that

186

α-LA treated by different ultrasonic powers combined with glycation gave a higher

187

IC50 value than native α-LA and untreated samples. It implies glycation can reduce the

188

IgG/IgE-binding abilities of α-LA, and ultrasonic pretreatment promoted the

189

reduction. The decrease in IgG/IgE-binding abilities of α-LA due to partial shielding



190

of some linear epitopes by conjugation with reducing galactose21, 25, and the structural

191

changes induced by glycation. Previous work reported that secondary and tertiary

192

structure of α-LA was disrupted by ultrasonication20, and ultrasonication can increase

193

glycation site(s) of α-LA and BSA16, 23. Yang et al. reported that ultrasonic treatment

194

combined with glycation can reduce the IgE/IgG-binding abilities of β-Lactoglobulin

195

by increase in glycation extent26. As show in the Fig. 2, the decreased

196

IgE/IgG-binding ability of α-LA under ultrasonication combined with glycation

197

treatment was also observed. Therefore, ultrasonic treatment combined with glycation

198

can also reduce the IgE/IgG-binding ability of α-LA by the increase in the glycation

199

extent and disrupt the structure. In order to understand their relationship clearly, the

200

glycation extent (illustrated by IR value, the number and location of glycation site,

201

and DSP value) were investigated using mass spectrometry in the subsequent

202

experiments.

203

Incorporation ratio (IR) of Gal to α-LA

204

Fig. 3 presents mass spectrometry analysis of all glycated samples. Average

205

molecular weights of N-LA-G, U-(LA-G)-90, U-(LA-G)-150, U-LA-G-90 and

206

U-LA-G-150 shifted to 14987.07, 15284.94, 15309.74, 15153.45 and 15168.48 Da,

207

respectively, much higher than the mass of the polypeptide backbone of α-LA,

208

14186.06 Da. Their IR was respectively calculated as 4.94, 6.78, 6.92, 5.97, 6.06.

209

This finding demonstrated that ultrasonic treatment significantly improved the

210

glycation degree of α-LA. However, MALDI TOF-MS can only measure intact


Page 10 of 34

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211

molecular weight of the treated α-LA, the location and number of glycation sites and

212

DSP value could be not revealed. To fully investigate them, HPLC-ETD MS/MS was

213

performed after pepsin digestion.

214

The location and number of glycation sites determination

215

If a peptide was glycated by galactose, and corresponding m/z of peak with the

216

charges of 1, 2, 3, 4, or 5 will appear mass shift 162.0528, 81.0264, 54.0176, 40.5132,

217

or 32.4106 accordingly. The results of mass spectrum analysis are shown in Fig. 4.

218

The m/z peaks of non-glycated peptide 9-23, 10-23, 53-71, 92-103, and 104-117 were

219

561.31393+, 512.29063+, 587.02334+, 463.94373+, 566.95903+, whereas the relative m/z

220

peaks of glycated peptide were 615.33143+, 566.30823+, 668.04974+, 571.98863+,

221

620.97673+, separately. The m/z shift of these peaks were 54.0175, 54.0176, 81.0264,

222

108.0449, and 54.0177 Da separately. This result indicated that these peptides had

223

mono-glycated, or dual-glycated peptides.

224

As we known, there are 12 Lys, 1 Arg and N-terminal of Asn45 in the native

225

α-LA, whence α-LA includes 13 potential glycation sites. We used HPLC-ETD

226

MS/MS to obtain the detailed map of glycation site K13, K16, K58, K62, K93, K98,

227

and K108 of α-LA (Fig. 5). Fig. 5A presents ETD MS/MS spectrum of

228

mono-glycated peptide 9FRELKDLKGYGGVSL23 with a peak at m/z of 615.33143+.

229

K13 was obtained by the mass difference between the c4 and c6 ions, or between the

230

z10 and z12, confirming that Gal was attached to K13. K16, K98 and K108 were also

231

determined by mass spectrometry of glycated peptide with m/z of 566.30823+,



Page 12 of 34

232

428.73764+, 620.97673+, respectively. Fig. 5B, 5D and 5F presents the c and z ions

233

were

234

91

235

104

236

5E

237

53

238

with m/z of 668.04974+, 571.98863+ were identified to be K58, K62, K93 and K98

239

respectively. These results indicated that HPLC-ETD MS/MS can identify the

240

location of glycation sites of α-LA clearly.

highly

matched

with

the

10

peptides

RELKDLKGYGGVSL23,

C(carbamidomethyl)VKKILDKVGINY103

and

WLAHKALC(carbamidomethyl)SEKLDQ117, respectively. Similarly, Fig. 5C and presents

ETD

MS/MS

of

the

FQINNKIWC(carbamidomethyl)KDDQNPHSSN71

dual-glycated and

92

peptides

VKKILDKVGINY103

241

Glycated peptide and glycation sites of N-LA-G, U-(LA-G)-90, U-(LA-G)-150,

242

U-LA-G-90 and U-LA-G-150 was summarized in Table 1. N-LA-G contains five

243

glycation sites, including K13, K16, K93, K98 and K108. We used ultrasonication at

244

90 and 150 W/cm2 to pretreat α-LA, six sites (K13, K16, K62, K93, K98, and K108)

245

were glycated. However, the mixtures of α-LA and Gal were treated at same

246

ultrasonic conditions, two additional glycated sites (K58 and K62) were found to be

247

glycated. The results were consistent with average molecular weight and IR value of

248

the treated α-LA (Fig. 3). The increase of glycation sites was probably because

249

structure of α-LA loosened with ultrasonic treatment, which accelerated glycation,

250

and exposed more reactive sites16, 27, 28. In present study, K58 was not observed in

251

U-LA-G-90 and U-LA-G-150, but it was detected in the U-(LA-G)-90 and

252

U-(LA-G)-150. Intriguingly, no obvious difference existed between the glycation site

253

of U-LA-G-90 and U-LA-G-150, the same result was found in the U-(LA-G)-90 and


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254

U-(LA-G)-150, suggesting that ultrasonic pretreatment can change the structure of

255

α-LA, confirmation of Gal and promote the glycation reaction between α-LA and Gal

256

as well. These results indicated that glycation sites were associated with the

257

pre-treated α-LA and galactose by ultrasonication. Fig. 6 shows that two additional

258

sites (K58 and K62) were found in the treated α-LA after ultrasonic treatment

259

combined with glycation. A very similar result was exhibited by the previous study,

260

where both glycated α-LA and BSA mainly occurred on Lysine29, 16. Therefore, it

261

seems credible that the number of glycation site can be affected by conformational

262

changes of α-lactalbumin treated by ultrasonic treatment.

263

Ultrasonication on the DSP value of α-LA

264

Fig. 7 shows the DSP value for all glycated peptides of N-LA-G, U-(LA-G)-90,

265

U-(LA-G)-150, U-LA-G-90 and U-LA-G-150. After ultrasonic treatment, glycated

266

peptide exhibited a higher DSP than that of un-treated samples. For example, K13 is

267

the reactive Gal glycation sites in N-LA-G with DSP value close to 0.19, however,

268

ultrasonic treatment increased their DSP value to 0.37, 0.64, 0.34 and 0.5 in the

269

U-(LA-G)-90, U-(LA-G)-150, U LA-G-90 and U-LA-G-150 respectively. Similarly,

270

the DSP value of other glycated peptides were increased by ultrasonic treatment. This

271

is because the structural change of α-LA by ultrasonic treatment, thus, gaining more

272

accessibility to the glycation, as had been previously demonstrated16. Interestingly, we

273

found the peptides of U-(LA-G)-90 had a higher DSP value compared to that of

274

U-LA-G-90, the similar phenomenon was observed between U-(LA-G)-150 and



275

U-LA-G-150. It was likely that α-LA and Gal were treated by ultrasonication, maybe

276

changed their conformation, and finally caused the increase in the glycation between

277

α-LA and Gal.

278

Analysis of ultrasonic pretreatment combined with glycation reduced

279

the IgE/IgG-binding abilities of α-LA

280

It is important to note that extent of Maillard reaction play an essential role in

281

explaining functional behaviors of Maillard reaction products (MRPs). In our recent

282

study, combining ultrasonic pretreatment with glycation observably reduced the

283

IgG/IgE-binding ability of α-LA, which was closely related to shielding effect of the

284

linear epitopes, the location of glycation sites, IR and DSP value. To explore this

285

result, high-resolution mass spectrometry can be employed to study the modification

286

of acid amino sequence, the location of glycation sites, and DSP value.

287

In the glycation reaction, saccharides can affect protein allergy, mainly by

288

masking the linear epitopes25. Järvinen et al.30 found that potential α-LA allergenic

289

epitopes identified are the fragments of the peptide 1-16, 13-26, 47-58, and 93-102.

290

These sequences contain one or more lysine residues (K13, K16, K58, K93, K94 and

291

K98), the glycation reaction between Lys and Gal results in modification of linear

292

epitopes with significant reduction of IgE/IgG reactivity. In this study, without

293

ultrasonic treatment, four sites (K13, K16, K93 and K98) were glycated in the

294

glycation between native α-LA and Gal, and results in linear epitopes changes, finally

295

led to a higher IC50 value compared to un-glycated samples (Fig. 2). As shown in


Page 14 of 34

Page 15 of 34


296

Table 1 and Fig. 6, K13, K16, K93 and K98 were discovered in all the glycated

297

samples, indicating that these epitopes were necessary to reduce the IgE/IgG-binding

298

ability of α-LA. It can be explained by the glycation of K13, K16, K93 and K98 sites

299

can mask the epitopic areas of α-LA. We applied ultrasonication at 90 and 150 W/cm2

300

to pretreat α-LA, one additional glycated site (K62) of glycated α-LA was found (Fig.

301

6). Although the epitopic areas of α-LA does not include K62, it can also alter acid

302

amino sequence of α-LA, and may be related to the IgE/IgG-binding ability. When the

303

mixtures of α-LA and Gal were ultrasonicated under same conditions, two additional

304

glycated sites K58 and K62 were identified (Fig. 6), which led to the conformation of

305

the linear epitopes changes, enhancing the IC50 value (Fig. 2), finally decreasing the

306

IgE/IgG-binding ability of α-LA. This suggests that high intensity ultrasonication uses

307

can bring about conformational changes of α-LA and Gal31, 32, improved the glycation

308

reaction and glycation sites, thereby decreased the IgE/IgG-binding ability. By

309

comparing IC50 value of all samples, glycation site K58 was identified in the

310

U-(LA-G)-90 and U-(LA-G)-150, which the IgE/IgG-binding ability reductions were

311

higher than that of U-LA-G-90 and U-LA-G-150. A recent study by Zhang et al.21

312

reported that glycation of K58 was dominant in α-LA-glucose, which the decreased

313

antigenicity was the highest. These findings indicated that the location and number of

314

glycation sites can significantly affect the IgE/IgG-binding ability of α-LA.

315

Although U-(LA-G)-90 and U-(LA-G)-150 had same number of glycation sites

316

(Table 1), U-(LA-G)-150 had a higher IR, DSP and IC50 value compared to those of

317

U-(LA)-G-90. The similar result was observed between U-LA-G-150 and U-LA-G-90



318

(Fig. 2, Fig. 3 and Fig. 7). Thus, α-LA underwent a reaction regions exposure after

319

ultrasonication treatment, these reaction regions were well exposed with increased

320

ultrasonic power, which improved the glycation extent of α-LA, eventually reduced

321

its potential allergenicity. These results indicated that the IgE/IgG-binding abilities of

322

glycated α-lactalbumin could be affected by the IR and DSP values. Additionally, the

323

structure of α-LA was changed by heat treatment, leading to the disruption of linear

324

epitopes33, finally impacting the IgE/IgG-binding ability of α-LA. Therefore,

325

ultrasonic treatment coupled with glycation decreased IgE/IgG-binding ability of

326

α-LA by both masking the epitopes, and by improving its glycation extent.

327

Conclusions

328

In this paper, combining ultrasonic pretreatment with glycation caused much

329

greater reduction in IgE/IgG-binding abilities of alpha-lactalbumin compared to the

330

individual dry-sate glycation alone. The result was attributed to the glycation of K13,

331

K16, K58, K93 and K98 sites can mask the linear epitopes of α-LA. Furthermore,

332

ultrasonic treatment promoted the reduction in IgE/IgG-binding ability of α-LA by

333

increasing the amount of glycation sites, IR and DSP value. However, some

334

experiments in vivo and vitro, such as double-blind placebo-controlled trial, and cells

335

experiment, need be measured to fully ensure the reduced allergenicity of glycated

336

α-LA during food processing.

337

Acknowledgements


Page 16 of 34

Page 17 of 34


338

This work was supported by the earmarked fund for China Agriculture Research

339

System

340

(20162BCB23017) and Chinese National Natural Science Foundation (No. 31460395

341

and 31760440).

342

Abbreviations

343

α-LA, alpha-lactalbumin; IgE, immunoglobulin E; IgG, immunoglobulin G; Gal,

344

Galactose; DSP, the degree of substitution per peptide; ELISA, enzyme-linked

345

immunosorbent assay; AA, amino acid; MALDI TOF, matrix-assisted laser desorption

346

ionization time of flight; HPLC, high performance liquid chromatography; ETD

347

MS/MS, electron transfer dissociation mass spectrometry/mass spectrometry.

348

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(CARS-45),

Excellent

Youth

Foundation

of

Jiangxi

Province

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Page 22 of 34

Page 23 of 34


Figure captions Fig. 1: Schematic depiction of the sample preparation. Fig. 2: The IgE (A) and IgG (B) binding ability of the treated α-LA was performed by inhibition ELISA. IC50: the concentration of inhibitors that causes a 50% inhibition of antibody binding (μg/mL). Pooled rabbit anti-α-LA-sera or Anti-α-LA patients' pooled sera (50 μL/well) were incubated separately with 0.5, 1, 5, 30, 60, 100 μg/mL (50 μL/well) of glycated α-LA as inhibitors. Fig. 3: MALDI-TOF-MS analysis of N-LA-G, U-(LA-G)-90, U-(LA-G)-150, U-LAG-90 and U-LA-G-150. Fig. 4: Mass spectra for the unglycated peptides of U-(LA-G)-150. (A) peptide 9-23 at +

+

m/z 561.31393 , (B) peptide 10-23 at m/z 512.29063 , (C) peptide 53-71 at m/z 587.02334+, (D) peptide 92-103 at m/z 463.94373+, (E) peptide 104-117 at m/z 566.95903+. The determined peptides are labelled by residue numbers. The m/z differences between glycated and unglycated peptides are indicated above the arrows. Fig. 5: The ETD MS/MS spectra of the glycated peptides. (A) the glycated peptide 923 (FRELKDLKGYGGVSL) with m/z of 615.33143+, (B) the glycated peptide 10-23 +

(RELKDLKGYGGVSL) with m/z of 566.30823 , (C) the glycated peptide 53-71 (FQINNKIWCKDDQNPHSSN) with m/z of 668.04974+, (D) the glycated peptide 91103 (VKKILDKVGINY) with m/z of 428.73764+, (E) the glycated peptide 92-103 (VKKILDKVGINY) with m/z of 571.98863+, (F) the glycated peptide 104-117 (WLAHKALCSEKLDQ) with m/z of 620.97673+. The sequence of per peptide is depicted on the top of the spectrum. The identified glycated sites are indicated by a



line with galactose. The c and z ions are shown by the numbers and lines. Fig. 6: Ribbon diagram of the glycated α-LA (PDB 1F6S). The glycation sites are colored as follows: grey, framework of α-LA; red, glycation sites of the glycated αLA; green, additional glycation sites of the glycated α-LA after ultrasonication. Fig. 7: The average degree of substitution per peptide molecule (DSP) value of glycated sites of N-LA-G, U-(LA-G)-90, U-(LA-G)-150, U-LA-G-90 and U-LA-G150.


Page 24 of 34

Page 25 of 34


Fig. 1: Schematic depiction of the sample preparation.



Page 26 of 34

Fig. 2: The IgE (A) and IgG (B) binding ability of the treated α-LA was performed by inhibition ELISA. IC50: the concentration of inhibitors that causes a 50% inhibition of antibody binding (μg/mL). Anti-α-LA rabbit pooled sera or Anti-α-LA patients' pooled sera (50 μL/well) were incubated separately with 0.5, 1, 5, 30, 60, 100 μg/mL (50 μL/well) of the corresponding glycated α-LA as inhibitors.

100

A

90

Inhibition (1-B/B0)*100

80 70

N-LA N-LA-G U-(LA-G)-90 U-(LA-G)-150 U-LA-G-90 U-LA-G-150

12.2

60

IC50

50

4.5

11.1

40

14.2 16.2 14.5

30 20 10 0 -0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Log(inhibitor concentration)

100

B

90

Inhibitor (1-B/B0)*100

80 70 60 50

N-LA N-LA-G U-(LA-G)-90 U-(LA-G)-150 U-LA-G-90 U-LA-G-150

3.9

1.1

IC50

4.6

5.1 6.4

5.5

40 30 20 10 0 -0.5

0.0

0.5

1.0

1.5

2.0

Log(inhibitor concentration)


2.5

3.0

Page 27 of 34


Fig. 3: MALDI-TOF-MS analysis of N-LA-G, U-(LA-G)-90, U-(LA-G)-150, U-LAG-90, U-LA-G-150.

100 100

100

U-(LA-G)-90

U-(LA-G)-150

15309.74

N-LA-G 14987.07

80

80

15284.94

40

60

40

20000

25000

30000

35000

0 10000

40000

15000

20000

25000

mass (m/z) 100

40

30000

35000

40000

0 10000

15000

20000

100

U-LA-G-90

15153.45

U-LA-G-150 15168.48

80

80

60

40

0 10000

60

40

20

20

15000

20000

25000

mass (m/z)

30000

35000

40000

0 10000

25000

mass (m/z)

mass (m/z)

% intensity

15000

60

20

20

20

0 10000

% intensity

% intensity

60

% intensity

% intensity

80

15000

20000

25000

30000

mass (m/z)


35000

40000

30000

35000

40000


Page 28 of 34

Fig. 4: Mass spectra for the unglycated peptides of U-(LA-G)-150. (A) peptide 9-23 at m/z 561.31393+, (B) peptide 10-23 at m/z 512.29063+, (C) peptide 53-71 at m/z 587.02334+, (D) peptide 92-103 at m/z 463.94373+, (E) peptide 104-117 at m/z 566.95903+ The determined peptides are labelled by residue numbers. The m/z differences between glycated and unglycated peptides are indicated above the arrows.

A

100

AA (9-23)

100

m/z=54.0175

20

0 550

560

570

580

590

600

+3 615.3314

610

620

60

40

m/z=54.0176 +3 512.2906

20

630

640

0 480

650

490

500

510

520

mass (m/z) 100

+3 566.3082

530

100

D

80

20

550

560

570

40

+4 627.5363

+4 587.0233

+3 571.9886 +3 517.9707 +3 m/z=54.027 463.9437

m/z=54.0179

m/z=40.513

580

0 560 570 580 590 600 610 620 630 640 650 660 670 680

mass (m/z) AA (104-117)

E

60

40

m/z=54.0177 20

0 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600

mass (m/z)

0 550

+3 566.959

560

570

580

+4 668.0497

m/z=40.5134

20

80

60

40

540

60

mass (m/z) AA (92-103)

Relative abundance

+3 561.3139

AA (53-71)

C

80

relative abundance

Relative abundance

60

40

100

80

Relative abundance

Relative abundance

80

AA (10-23)

B

590

600

610

mass (m/z)


+3 620.9767

620

630

640

650

Page 29 of 34


Fig. 5: The ETD MS/MS spectra of the glycated peptides. (A) the glycated peptide 923 (FRELKDLKGYGGVSL) with m/z of 615.33143+, (B) the glycated peptide 10-23 (RELKDLKGYGGVSL) with m/z of 566.30823+, (C) the glycated peptide 53-71 (FQINNKIWCKDDQNPHSSN) with m/z of 668.04974+, (D) the glycated peptide 91-103 (VKKILDKVGINY) with m/z of 428.73764+, (E) the glycated peptide 92-103 (VKKILDKVGINY) with m/z of 571.98863+, (F) the glycated peptide 104-117 (WLAHKALCSEKLDQ) with m/z of 620.97673+. The sequence of per peptide is depicted on the top of the spectrum. The identified glycated sites are indicated by a line with galactose. The c and z ions are shown by the numbers and lines. AA (9-23) C

2 3 4 5 6 7 8 9 10 11 12 13 14 15

AA (10-23)

F R E L K D LK GY G GX S L

A

15 14 13 12

100

c

B

Z

10 9 8 7 6 5 4 3 2

Gal

926.4910,z8

1039.5513,z9 1062.5466,c7 1119.6566,c8 1154.5414,z10 1282.7168,c9,z11 1339.7493,c10 1396.7416,c11,z+1 12 1495.7174,c12 1524.7667,z13 1582.8691,c13

772.5216,c6

544.4542,c4 579.2954,z6 636.8537,z7 659.4356,c5

40

303.1801,c2 360.2628,z+1 4 416.4059,c3,z5

Relative abundance

1730.8760,c+1 14

1681.9294,z+1 14

60

20

0

0 600

800

200

1000 1200 1400 1600 1800 2000

400

600

800

1000

AA (53-71)

20

100 Z

Gal 80

0

1400

1600

1800

AA (91-103) D

c

60

40

20

2 3 4 5

6 7

8 9 10 11 12 13

C VKK I LDK VG I NY 13 12 11 10 9

8 7

5

4 3

2

Gal 1067.5203,z8

639.4443,z6

3 2

Relative abundance

40

767.4174,z7

60

293.3460,c2 406.3763,c3 428.3430,z4

Relative abundance

80

634.5757,c5

520.4766,c4

Gal

9 8 7 6 5 4

1633.7390,z12 1673.3113,c10 1747.7017,z+1 13 1789.8377,c+1 11 1903.8579,c12

924.6260,c6 997.4681,z9 1016.8621,c132+ 2+ 1037.6895,c7 1076.3084,z15 1122.3626,c152+ 2+ 1190.4365,z17 ,c162+ 1223.7468,c8 1234.7122,c172+ 1254.7854,z182+ 2+ 1289.7769,z+1 10 1278.7280,c18 1383.7105,c9 1447.7351,z11

13 12 11

759.5822,c6 838.5655,z6 874.5732,c7 954.4459,z7

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

646.4925,c5

2 3

R Q I N N K I WC KD DQ N P H S S N 19 18 17 16 15

277.1972,c2 280.0900,z2 393.2735,z3 405.3481,c3 450.3312,z4 549.4022,z5 533.4385,c4

C

C

1200

m/z

m/z

1309.7411,z10+1

400

1321.7047,c10+1 1433.8119,c11 1437.8254,z11+1 1536.8190,z12+1

200

100

7 6 5 4 3 2

14 13121110 9

80

174.1142,c1

20

764.4419,z8 853.5383,c5 877.5728,z9 968.4913,c6 992.5053,z10 1081.5833,c7 1209.6196,c8

321.2676,c2 450.2798,c3

563.3777.c4 638.9337,z7

40

416.1037,z5

Relative abundance

60

1226.5387,c9 1282.7128,z11 1395.7565,z12 1429.7710,c10 1486.7706,c11 1524.7389,z13 1543.8335,c12 1643.8618,c+1 13

Gal 80

2 3 4 5 6 7 8 9 10 1112 13 14

R E L K D L KGYGGVS L

1164.6488,c8 1180.5967,z9 1263.7186,c9

100

1200

1400

0 200

400

600

800

1000 1200 1400 1600 1800 2000

200

400

600

800

m/z

1000

m/z


1600

1800

z

z

40

20

200 400 600 800 1000 1200

Gal

0 1400 1600 1800 60

40

246.3444,z2

100

z

80

20

0 200 400 600

815.5446,c5 886.6234,c6 866.7630,z7 977.4742,z8 999.6346,c7 1048.4681,z9 1160.5671,c8 1247.6023,c9 1338.6513,z10 1376.6639,c10 1475.6777,z11 1504.7372,c11 1546.7170,z12 1617.8076,c12 1659.8136,z13 1732.0287,c13

AA (92-103) 359.3906,z3 388.4573,c3 487.4854,z4 525.4684,c4 617.4882,z+1 5 703.4441,z6

2 3 4 5 6 7 8 9 10 11 12

204.3369,c1

V K K I LD K V G I N Y

317.4932,c2

5 4 3 2

Relative abundance

10 9 8 7

1598.8585,z11

Gal 1323.7513,c+1 9

12

1435.7590,c10

80

1549.9285,c11

c

1166.1193,c7 1180.7336,z9 1265.7496,c8 1308.6963,z10

E

1067.6547,z8

761.6832,c5 839.3754,z6 876.7221,c6 954.3035,z7

407.4978,c2

60

451.5798,z+1 4 535.3893,c3 549.6426,z5 648.4789,c4

100

393.6325,z3

Relative abundance


F C

m/z 800

m/z


Page 30 of 34

W L AH K AL CS E K LD Q

2 3 4 5 6 7 8 9 10 11 12 13 14

AA (104-117)

14 13 12 11

Gal

9 8 7 6 5 4 3 2

Z

0 1000 1200 1400 1600 1800 2000

Page 31 of 34


Fig. 6: Ribbon diagram of the glycated α-LA (PDB 1F6S). The glycation sites are colored as follows: grey, framework of α-LA; red, glycation sites of the glycated αLA; green, additional glycation sites of the glycated α-LA after ultrasonication.



Page 32 of 34

Fig. 7: The average degree of substitution per peptide molecule (DSP) value of glycated sites of N-LA-G, U-(LA-G)-90, U-(LA-G)-150, U-LAG-90 and U-LA-G-150. N-LA-G U-(LA-G)-90 U-(LA-G)-150 U-LA-G-90 U-LA-G-150

1.0

0.8

DSP

0.6

0.4

0.2

0.0 9-23

10-23

53-71

92-103

peptide


104-117

Page 33 of 34


Table 1. Summary of the glycated peptides in the N-LA-G, U-(LA-G)-90, U-(LA-G)-150, U-LA-G-90 and U-LA-G-150. Sample N-LA-G

Peptide

m/z

Δm

location

Glycated peptide

ppm

9-23

615.33053+

-1.68

10-23

566.30903+

91-103

428.73774+

92-103

571.98903+

104-117

Sequencea

Glycated

(V)FRELKDLKGYGGVSL(P)

K13

0.53

(F)RELKDLKGYGGVSL(P)

K16

-0.23

(M)C*VKKILDKVGINY(W)

K98

-6.01

(C)VKKILDKVGINY(W)

K93, K98

465.98484+

0.48

(Y)WLAHKALC*SEKLDQ(W)

K108 K13

site

U-(LA-G)-90 9-23

615.33143+

-0.16


10-23

566.30873+

0.06


K16

53-71

668.04964+

-0.68

(L)FQINNKIWC*KDDQNPHSSN(I)

K58, K62

91-103

428.73804+

0.53


K98

92-103

571.98893+

-6.13

(C)VKKILDKVGINY(W)

K93, K98

104-117

620.97723+

0.91


K108 K13

U-(LA-G)-150 9-23

615.33143+

-1.52


10-23

566.30823+

0.71


K16

53-71

668.04974+

-1.04


K58, K62

91-103

428.73764+

-0.47


K98

92-103

571.98863+

-6.13

(C)VKKILDKVGINY(W)

K93, K98

104-117

620.97673+

1.07


K108

9-23

461.75074+

0.49


K13

10-23

566.30923+

0.88


K16

53-71

627.53694+

0.40


K62

91-103

571.31543+

1.34


K98

92-103

571.98933+

-5.60

(C)VKKILDKVGINY(W)

K93, K98

104-117

465.98364+

-1.61


K108

9-23

615.33153+

-0.05


K13

10-23

566.30873+

-0.12


K16

53-71

627.53684+

0.20


K62

91-103

428.73784+

0.18


K98

92-103

571.98883+

-6.36

(C)VKKILDKVGINY(W)

K93, K98

104-117

465.98484+

0.56


K108

U-LA-G-90

U-LA-G-150

aC*

refers to carbamidomethyl.



Page 34 of 34

Graphical abstract

Native α-LA Dry-heating glycation with galactose

Ultrasonic pretreatment D-(+)-Galactose

Ultrasonic pretreatment Dry-heating Lys 108

Lys 108 Lys 16

Lys 13

Lys 16 Lys 98 Lys 93

Dry-heating glycation with galactose

Lys 13

Lys 108 Lys 16

Lys 98 Lys 93 Lys 58

Lys 62 Glycation extent ACS Paragon Plus Environment IgE/IgG binding ability

Lys 13

Lys 98 Lys 93

Lys 62

Fluorescence of Fluorescence of Cyclopropenium Ion

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