N-Glycosylation Plays An Essential and Species-Specific Role in Anti

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Functional Structure/Activity Relationships

N-Glycosylation Plays An Essential and SpeciesSpecific Role in Anti-Infection Function of Milk Proteins Using Listeria monocytogenes as the Model Pathogen Feng Zheng, Yamin Du, Xisha Lin, Liqi Zhou, Yun Bai, Xiaobo Yu, Josef Voglmeir, and Li Liu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 03 Sep 2019 Downloaded from pubs.acs.org on September 3, 2019

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Journal of Agricultural and Food Chemistry

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N-Glycosylation Plays An Essential and Species-Specific Role in Anti-

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Infection Function of Milk Proteins Using Listeria monocytogenes as

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Model Pathogen

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Feng Zheng1†, Ya M. Du2†, Xi S. Lin1, Li Q. Zhou1, Yun Bai3, Xiao B. Yu3, Josef Voglmeir1*,

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Li Liu1*

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Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food

8

1

9

Science and Technology, Nanjing Agricultural University, Nanjing, China School of Food Science and Engineering, Qilu University of Technology (Shandong

10

2

11

Academy of Science), Jinan, China

12

3

13

Nanjing, China

14

†

National Center of Meat Quality and Safety Control, Nanjing Agricultural University,

The authors equally contributed to this work.

15 16

*Correspondence

should be addressed to:

17

E-mail: [email protected], Fax: +86 25 84399553 Tel: +86 25 84399512 or

18

E-mail: [email protected]: Fax: +86 25 84399553 Tel: +86 25 84399512

19 20

Keywords: milk N-glycosylation; anti-pathogenic function; glycoprotein; Listeria

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monocytogenes

1

ACS Paragon Plus Environment


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Abstract: The released milk N-glycome has been found to possess anti-

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pathogenic activity. Natively, they are covalently linked onto proteins. Whether

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the conjugated N-glycans still have anti-pathogenic properties, and how the

25

glycosylation influences the anti-pathogenic activity of proteins remains

26

unclear. Herein we compared the quantitative differences of milk protein N-

27

glycosylation, and the anti-listerial differences of native milk proteins, released

28

N-glycan pools, and de-glycosylated proteins between human and bovine milk.

29

N-glycosylation exhibited to be quantitatively species-specific. The entire

30

growth inhibitory activity and the majority of the anti-adhesive activity against

31

L. monocytogenes of milk whey proteins, although not as high as the released N-

32

glycans, are attributed to N-glycosylation. Moreover, all N-glycan-bearing

33

samples from human milk showed better growth inhibitory activities than

34

those from bovine milk. Generally, N-glycosylation significantly contributes to

35

the anti-listerial function of milk proteins and to the functional differences

36

between species. This gives novel insights into the role of these glycoconjugates

37

in nature.

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Introduction

39

Mother milk is the exclusive feed source for newborns in all mammals, as it

40

contains all nutrients and bioactive components essential for the healthy

41

development of infants.(1) Numerous studies have been carried out to explore

42

the biological functions of different milk components.(2-5) The oligosaccharide

43

portion in milk has also drawn great attention of researchers regarding their

44

anti-pathogenic functions towards different pathogens, and are believed to be

45

the major barrier for pathogen invasion in the gut.(6-8) However, the majority of

46

these studies are focused on free oligosaccharides in milk. A rather large

47

proportion of the milk proteins contain conjugated oligosaccharide chains

48

linked to asparagine (so-called N-glycans), which function as a food component

49

has not yet been extensively studied.

50

We have previously reported that the N-linked glycans released from both

51

human and bovine milk exhibited obvious anti-pathogenic activities, which

52

include both growth inhibition of the pathogen and the anti-adhesion of the

53

pathogen to Caco-2 intestine cells.(9) This was the first report about the growth

54

inhibitory effect of human milk N-glycans on pathogens, and shed light onto

55

novel biological functions of human milk. However, this function was from the

56

freed N-linked glycans enzymatically released from milk glycoproteins. N-

57

glycans in nature are attached to the proteins in milk when consumed, and

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whether the protein-conjugated N-glycans have the same function as the

59

released N-glycans still remains unanswered.

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Glycosylation confers various in vivo functions, such as signal conduction, cell-

61

cell recognition, and immune regulation, to proteins.(10-14) These described

62

functions are all associated with the cellular functions of N-glycans in health

63

and disease. However, the function of protein N-glycosylation in exogenous

64

substances such as in food remains unknown.

65

The biological functions of milk glycoproteins have been extensively

66

documented. For instance, lactoferrin was reported to be able to protect the

67

intestinal cells against viral and bacterial infections, stimulates immune cells,

68

and to inhibit the production of pro-inflammatory molecules.(15-17) However,

69

whether these functions are attributed to the protein backbone or to the

70

attached N-glycans was not elucidated in detail. It was reported that the

71

activities of lactoferrin against Salmonella enterica and E. coli O157:H7 changed

72

when it was treated with exo-glycosidases,(18) indicating the apparent role of

73

the sugar moieties in the anti-pathogenic function of this glycoprotein.

74

Moreover, the anti-influenza A virus activity of bovine milk proteins containing

75

Sia (sialic acid) α2-3/6Gal (galactose)-linked glycans disappeared when the

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terminal sialic acid moieties were removed.(19) N-glycosylation of hmLF

77

(human milk lactoferrin) significantly inhibited pathogen adhesion, and the

78

purified glycans from hmLF significantly reduced the invasion of colonic

79

epithelial cells by Listeria to levels similar to non-invasive mutants.(18) These

80

findings indicate that N-glycans also play roles in anti-pathogenic functions

81

when linked to proteins.

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Listeria monocytogenes is a common pathogen which is the etiologic agent of

83

listeriosis, a severe foodborne disease leading to blood and brain infections in

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humans and many animal species.(20, 21) Infections during pregnancy can lead

85

to severe complications and infection of the newborn.(22-24) It is therefore of

86

great importance to discover an effective and safe approach to inhibit or kill

87

this pathogen. L. monocytogens was one of the pathogens sensitive towards

88

human and bovine milk N-glycans in our previous study. Here we used this

89

bacterium as the target pathogen to further investigate the anti-listerial activity

90

of the N-glycome, the native proteome and the de-glycosylated proteome

91

samples from human and bovine milk, in order to better understand the anti-

92

listerial activity of milk N-glycans.

93 94

Materials and methods

95

Milk samples collection and preparation

96

Bovine milk samples were collected from a local dairy farm (Xi-Gang Fruit

97

Ranch, Nanjing). Milk samples were taken from five cows at the time of

98

colostrum (before day 7), 1 month, 3 months and 6 months postpartum,

99

respectively. Human milk samples were provided by five healthy women

100

between 28 and 32 years of age and collected at the same lactation intervals as

101

described for the cow milk samples. All samples were kept in cooling bags at

102

4°C until their transfer to a -80°C freezer within three hours and stored there

103

until analysis.

104

Nitrogen analysis 5



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Total nitrogen and non-protein nitrogen were determined using a Kjeldahl

106

Analyzer (Foss TM 2300, Switzerland). In brief, 1 ml of milk was freeze-dried

107

and mixed with 10 ml H2SO4. One copper sulfate/titanium oxide catalyst tablet

108

(1 g, Shoude, Nanjing) was added and the mixture was digested at 420°C for 1

109

h. After the digestion, nitrogen was determined according to the

110

manufacturer’s instructions. For the determination of non-protein nitrogen,

111

the protein was precipitated by TCA (trichloroacetic acid, 12% w/v). The

112

nitrogen content in the supernatant was detected as the non-protein nitrogen.

113

Protein nitrogen was then calculated by subtracting the non-protein nitrogen

114

content from the total nitrogen content.

115

Preparation of native whey protein, de-glycosylated whey protein, and N-glycome

116

samples

117

The human and bovine milk samples were completely thawed and defatted

118

through the removal of the upper layer of fat after centrifugation at 13500 rpm

119

and 4°C for 30 min. The skimmed milk was dialyzed with 8000-14000 Da

120

dialysis bag (Shyuanye Company, Shanghai, China) on ice to remove free

121

oligosaccharide and other small molecules, and the UHPLC (Shimadzu Nexera,

122

Kyoto, Japan) was used to monitor the free oligosaccharides removal. 1 M HCl

123

was added into the oligosaccharide-free milk to adjust the pH to 4.5 and the

124

sample was centrifuged (8000 g, 20 min) to obtain the whey protein in the

125

supernatant. The obtained whey protein was then neutralized drop-wise with

126

1 M NH4HCO3 and used as the native milk whey protein sample.

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PNGase F (300 U, Qlyco Ltd., Nanjing, China) was then used for the protein de-

128

glycosylation according to the method described previously.(9) In brief, milk

129

was defatted by centrifugation at 5000 g for 20 min at 4°C. The lower phase

130

was mixed with an equal volume of TCA (40% w/v) and the resulting

131

suspension was further centrifuged at 5000 g for 40 min. The pellet was

132

washed with distilled water to remove lactose and free oligosaccharides from

133

the debris and the supernatant was neutralized with NaOH solution (0.1 M).

134

The resulting milk (glyco-)protein pellets were solubilized in 150 ml of urea

135

solution (6 M), followed by the addition of 120 ml of phosphate buffer (500

136

mM, pH 7.5), 60 ml of sodium dodecyl sulfate solution (2% w/v SDS in 1 M β-

137

mercaptoethanol) and 600 ml distilled water. The mixtures were boiled for 5

138

min and then cooled down. 100 ml of Triton X-100 solution (10% v/v) and 150

139

ml of PNGase F were added into the samples and incubated at 37°C for 16 h.

140

The released N-glycome sample was separated from the protein through

141

ultrafiltration (10 KDa, Shyuanye Company, Shanghai, China). The isolated N-

142

glycome preparation was then desalted and further purified using size

143

exclusion chromatography (Biorad P2 fine, in 1% (w/v) aqueous acetic acid

144

solution). Fractions containing carbohydrates (tested by sulfuric orcinol

145

staining) were pooled. To test the purity of the sample, ninhydrin staining was

146

performed to the pooled N-glycans to ensure that the sample is free of peptide

147

fragments or other amine sources. The sample retained in the ultrafilter after

148

the last centrifugal filtration was the de-glycosylated protein. The complete de-

149

glycosylation of whey protein was monitored by detecting the disappearance 7



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of N-glycans from the filtrate using UHPLC analysis. After a few washes by

151

distilled water, the de-glycosylated protein was used for further functional

152

tests.

153

Quantitive SDS-PAGE analysis of proteins

154

Gel imaging (Bio-Rad, USA) was used to quantify the glycoproteins in milk

155

based on the intensity of SDS-PAGE gel bands before and after PNGase

156

treatment. Whey protein samples were adjusted to a final protein

157

concentration of 1.0 μg/μl and heated at 95°C for 5 min. 25 μg proteins were

158

added to each lane. Proteins were stained with Coomassie blue R250 and de-

159

stained afterwards. The relative intensity of each band was quantified using the

160

Quantity One imaging software package (Bio-Rad, USA).

161

N-glycan quantification

162

The released N-glycans from milk protein were detected on UHPLC using a

163

previously described analysis method based on hydrophilic interaction liquid

164

chromatography (HILIC) and fluorescence detection.(25) The concentration of

165

N-glycans was calculated based on the UHPLC peak areas using an equal

166

volume of 2AB labeled commercial maltopentaose (10 μM) as the internal

167

standard

168

according

10 μM ∗ glycan peak area maltopentaose peak area

to

the

formula:

milk glycans concentration =

(1).

169

Fucosidase, sialidase, and mannosidase (Qlyco Ltd, Nanjing) were used to

170

remove the fucose, sialic acid and mannose residues from the N-glycome,

171

respectively, for the relative quantification of fucosylated, sialylated and high-

172

mannosylated types of N-glycans. 5 replicates from each of the 5 different 8


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individuals were included for each test. The percentage of each of the three

174

types of N-glycan was calculated based on the shift of UHPLC peak retention

175

times. Sample peaks which shifted after glycosidase treatment refer to the N-

176

glycans containing the corresponding monosugar. The percentages of the

177

shifted peak areas over the sum of all peak areas were taken as the relative

178

amount of the respective N-glycan.

179

Quantification of sialic acids in milk N-glycans

180

The purified N-glycans obtained from 200 μl skimmed bovine and human milk

181

were used as the substrates. Each sample was hydrolyzed for 3 h at 80°C in 1

182

ml of 2 M acetic acid, with 2 μl of Neu5Prop(26) (4 mM) being added as an

183

internal standard. The mixture was cooled down to room temperature. 800 µl

184

of supernatant was pipetted from each sample after centrifugation and dried.

185

The dried sample was re-dissolved in 100 µl of 0.1 M NaCl. After centrifugation,

186

50 µl supernatant was mixed with 20 µl of the OPD reagent (10 mg/ml O-

187

phenylenediamine in 0.2 M NaHSO3). Then all samples were heated at 80°C for

188

40 min in dark and subsequently subjected to HPLC analysis. Samples were run

189

on a Nexera LCMS 2020 system (Shimadzu, Kyoto, Japan) equipped with a

190

reversed-phase HPLC column (Phenomenex Hyperclone 5 μm ODS, 250 × 4.60

191

mm), at a flow rate of 1.0 ml/min. Sialic acid derivatives were separated and

192

eluted with a three-phase mobile system and detected by a fluorescent detector

193

(excitation 373 nm, emission 448 nm). Solvent A was water, solvent B was

194

acetonitrile and solvent C was methanol. 5% B and C were applied from 0 to 10

195

min and, were both increased to 40% over 2 min from 10 min to 12 min and 9



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held at 40% for 4 min. B and C were then decreased to 5% in 1 min, and the

197

column was equilibrated with the initial conditions for 5 min. The Neu5Ac and

198

Neu5Gc content of bovine and human milk were quantified according to the

199

formula:

200

Neu5Ac content (μg/ml) = Neu5Ac molar mass *

201

Gc content (μg/ml) = Neu5Gc molar mass *

202

Bacterial growth inhibition assay

203

Listeria monocytogenes ATCC 19115 was recovered at 37°C for 12 h in BHI

204

medium for 3 times prior to the growth test. The isolated N-glycome, the native

205

whey protein, and the de-glycosylated whey protein, each from 5 ml milk, were

206

filter-sterilized (0.22 μM nitrocellulose filter, Millipore) and then added into

207

the bacterial culture and incubated for 24 h. An N-glycan preparation using

208

heat-inactivated PNGase F was used as the negative control. The bacterial

209

growth was monitored by measuring the optical density of the culture at 600

210

nm (OD600) using a micro photometer (NanoDrop, Nanjing, China) in 2 h

211

intervals.

212

Anti-adhesion assay

213

Listeria monocytogenes ATCC 19115 cells were recovered as stated above. The

214

bacterial pellet was then washed with an equal volume of 0.01 M PBS buffer

215

(pH 7.2) for 3 times, and re-suspended in DMEM medium (without FBS and

216

antibiotic) to an OD600 value of 0.2. The Caco-2 cells (human colon carcinoma

217

cell line ATCC HTB-37) were routinely grown in 6 ml microwell dishes, using

8 nmol * Neu5Ac peak area Neu5Prop peak area

8 nmol * Neu5Gc peak area Neu5Prop peak area

(2) Neu5

(3)

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the cell culture in DMEM medium containing FBS, 1% penicillin and 1%

219

streptomycin, at 37°C in 5% CO2 atmosphere. After 2 days of incubation, the

220

Caco-2 cells were transferred into a 96-well plate and cultured until the bottom

221

surface was fully covered. The cells from each well were washed with 200 µl

222

0.01 M PBS (pH 7.2) for 3 times prior to the addition of milk samples and the

223

bacterium. The filter-sterilized N-glycans, the glycoproteins and the de-

224

glycosylated proteins (each derived from 5 ml milk), were mixed with the

225

bacterial resuspensions (each 50 µl), respectively. The mixtures were then

226

added into each well and incubated together with the cells at 37°C in 5% CO2

227

atmosphere for 90 min. After washing with 200 µl PBS buffer (0.01 M) for 5

228

times (to removethe non-adhered bacteria), the cells from each well were lysed

229

using 0.1% Triton- X100 (in PBS buffer, w/v) for 5 min with shaking. Serial

230

dilutions of the cell lysates were plated on the solid medium, and the CFU of L.

231

monocytogenes were counted after 12 h incubation at 37°C.

232

Statistical methods

233

For all the quantitative assays, the experiments were carried out in triplicates.

234

The result is given as the average with standard error. The statistical methods

235

used were one-way ANOVA and Duncan multiple comparison. Differences were

236

considered statistically significant when the p-values were less than 0.05.

237 238

Results

239

Total protein and N-glycome level in whole human and bovine milk

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For both human and bovine milk, samples from 5 donors were collected at the

241

same lactation stages and investigated. Generally, the colostrum milk contained

242

higher protein levels than milk from other lactation periods for both human

243

and bovine (Fig. 1A). The protein level decreased from 2.3% ± 0.7 before day 7

244

to 1.3% ± 0.3 after 6 months in human milk, with a significant decrease

245

occurring after 1 month. During the same lactation period in bovine milk, the

246

protein level decreased from 4.7% ± 1.1 to 3.3% ± 0.3 with the level of

247

colostrum being significantly higher than that at other lactation stages. No

248

significant difference of protein content was exhibited for samples taken at

249

later lactation stages.

250

The N-glycan level in milk generally decreased in later lactation stages. It was

251

reduced from 5.8 nmol/100 µl in colostrum to 2.1 nmol/100 µl in 6-month

252

postpartum milk for human samples, and from 2.3 nmol/100 µl to 0.7

253

nmol/100 µl for bovine samples (Fig. 1B). Noticeably, in contrast to the higher

254

protein content in bovine milk, the glycan level of bovine milk was significantly

255

lower than that of human milk at all tested time points, showing that the

256

absolute amount of N-glycans in human milk is higher compared to bovine

257

milk.

258

Gel image results showed that the content of glycoproteins in human milk was

259

higher than that in bovine milk. This is consistent with the result of N-glycan

260

levels in two species and explained, to a certain extent, why the N-glycan level

261

in human milk is higher, whereas the protein level is lower than that in bovine

262

milk (Table 1). However, the glycan level in milk does not only depend on the 12


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amount of N-glycosylated proteins but also the extent of glycosylation of

264

individual glycoproteins. The latter probably makes more contributions to the

265

difference in total N-glycans between human and bovine milk.

266

Level of fucosylated, sialylated and high-mannosylated N-glycan structures in milk

267

The terminal modification of N-glycans plays important roles in their biological

268

functions. Fucosylation, sialylation, and high-mannosylation are the three

269

types of terminal modification studied most extensively. Generally, the level of

270

fucosylated structures in human milk was significantly higher than that of

271

bovine milk at each tested time point. The relative abundance of fucosylated

272

structures in human milk varied between 53.4% and 48.9%, and no significant

273

difference was found between any two lactation stages. In bovine milk, the

274

abundance of fucosylated structure varied between 33.9% and 22.7% with a

275

slight decrease over the tested lactation period but no significant change was

276

shown (Fig. 3A). The level of sialylated structures varied between 37.1% and

277

24.3% in human milk, and between 37.9% and 31.3% in bovine milk. The

278

difference between human and bovine milk was not significant at any tested

279

stage, and no significant change between any two time points for either human

280

milk or bovine milk was observed (Fig. 3B). The level of high-mannose

281

structures in human milk was lower than that in bovine milk but the significant

282

differences were only seen at month 3. These concentrations varied from

283

18.3% to 11.6% in human milk and from 22.9% to 18.1% in bovine milk.

284

Similarly, no significant dynamic change with time was observed (Fig. 3C).

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In human milk, the level of fucosylated structures was significantly higher than

286

that of sialylated structures, and both were higher than that of the high-

287

mannose structures (fucosylated > sialylated > high-mannosylated). In bovine

288

milk, the level of sialylated N-glycans was slightly higher than that of

289

fucosylated structures, and both were higher than that of high-mannose type

290

structures (fucosylated ≈ sialylated > high-mannosylated).

291

Sialic acid (Neu5Ac/Neu5Gc) contents of human and bovine milk

292

Neu5Ac but no Neu5Gc was detected in human milk from any individual at any

293

lactation stage, however, both Neu5Gc and Neu5Ac residues were observed in

294

bovine milk, indicating that Neu5Gc is specific for bovine milk. Both Neu5Ac

295

and Neu5Gc concentrations significantly decreased after day 7 and stayed

296

constant afterwards. For Neu5Ac, the concentration decreased from 83.4

297

μg/ml before day 7 to 10.2 μg/ml after month 6 in human milk, and from 24.8

298

μg/ml to 10.9 μg/ml over the same lactation period in bovine milk. The Neu5Ac

299

content in human milk was significantly higher than that in bovine milk at the

300

colostrum and 1st-month samples. In bovine milk, the determined Neu5Gc level

301

was similar as that of the Neu5Ac and decreased from 15.7 μg/ml to 1.1 μg/ml

302

within the tested lactation period (Fig. 4).

303

Amounts of specific N-glycans present in both human and bovine milk

304

Based on our previous study, the N-glycomes from human and bovine milk are

305

significantly different in their structural composition, and detailed structures

306

were elucidated using UHPLC and MALDI-TOF-MS/MS analysis.(9) In this study,

307

the N-glycan structures which are commonly shared by both human and bovine 14


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milk were analyzed and quantitatively compared based on the obtained UHPLC

309

profiles. The results showed that, although structures are present in both

310

human and bovine milk, their quantities varied significantly (Table 2). In

311

general, all observed N-glycans, except for the M6 and A2 glycoforms, were

312

more abundant in human milk samples. The glycoforms A3, M5, A2G2, FA2G2,

313

and M7 were significantly higher in human milk than in bovine milk at all

314

lactation time points. The glycoform FA3G1 was in significantly higher

315

abundance and M6 in a significantly lower abundance in later lactation stages

316

in human milk. For quantitative analysis, only the 9 most abundant N-glycan

317

structures listed in Table 2 were included in the comparison of human and

318

bovine milk glycans, whereas less abundant N-glycan structures (consisting of

319

less than 1% of the total N-glycans) and the structures co-eluted with other

320

non-commonly shared N-glycans were not considered in this comparative

321

study.

322

Anti-pathogenic activity associated with protein glycosylation

323

The human and bovine NPs and the bovine milk NG had similar activity at the

324

stationary phase, which is higher than those of the two DP samples but lower

325

than that of the human milk N-glycome samples (Fig 5). The significance

326

analysis at different time points showed that all samples including the negative

327

control (milk N-glycans prepared using heat-inactivated PNGase F) exhibited

328

obvious inhibitory activities against L. monocytogenes at 8 h of bacterial growth

329

compared to the blank control (sample volume replaced by water). No

330

inhibitory activity was observed for both deglycosylated human milk whey 15



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331

proteins (hDP) and bovine milk whey proteins (bDP). Both NP samples and

332

both NG samples continued to show significant anti-pathogenic activities, with

333

the human N-glycome sample (hNG) showing the highest activity after 24 h

334

incubation time (Fig. 5). The lose of growth inhibitory effect of milk whey

335

proteins towards L. monocytogenes after deglycosylation indicated that N-

336

glycosylation is the main cause for the growth inhibitory function of milk whey

337

proteins. On the other hand, the samples which still had N-glycans covalently

338

attached to proteins showed significantly lower anti-pathogenic activity than

339

the released N-glycans. hNG showed throughout higher activity than bNG and

340

hNP over the whole tested period, and the activity of bNG gradually decreased

341

and became the same as bNP at the end of the bacterial growth study.

342

NP, DP, and NG samples, from both human and bovine milk, were also tested

343

for their anti-adhesion activity to the Caco-2 cells (Fig. 6). It was shown that all

344

samples, including the DP preparations, exhibited anti-adhesion activities

345

compared to the negative and blank controls. In detail, the two milk NG samples

346

had significantly higher activities than the two NP samples, and the two NP

347

samples had significantly higher activities than the two DP samples. Moreover,

348

no significant differences between any two same type of samples from human

349

and bovine milk was observed. It is clear that, similarly to the observed growth

350

inhibition activities, N-glycosylation plays a critical role in the anti-adhesion

351

activity of milk proteins, although the deglycosylated protein itself also has low

352

anti-adhesive activity when compared to the controls. Furthermore, the tested

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glycoprotein (NG) is not as effective as the released N-glycans with respect to

354

its anti-adhesion activity.

355 356

Discussion

357

In our previous study, we performed the anti-pathogenic activity test using

358

isolated milk N-glycans and found that these oligosaccharides exhibited strong

359

growth inhibition and anti-adhesion activities to several commonly occurring

360

pathogens. Moreover, isolated human milk N-glycans functioned better than

361

the bovine milk N-glycans.(9) This finding brought up the question of how these

362

N-glycans act when they are in their native form, which is attached onto

363

proteins, and how the N-glycosylation affects the activity of the carrier protein.

364

Although milk N-glycan structures have been reported extensively,(27) the

365

comparison of the extent of N-glycosylation and a comparative study on

366

functional features of N-glycosylation between human and bovine milk is still

367

missing. Moreover, no study on the effect of the whole N-glycosylation pool on,

368

and the functional contribution to, their carrier proteins has been conducted so

369

far, despite of the reported roles that special terminal sugar moieties play in

370

the anti-pathogenic activity of glycoproteins.(18, 19) Therefore, this study aims to

371

reveal certain quantitative features of protein N-glycosylation and the

372

associated anti-listerial activity of milk whey proteins from both human and

373

bovine milk.

374

Interestingly, human milk contains a much lower level of proteins but a much

375

higher level of N-glycans than bovine milk. This may be the result of natural 17



Page 18 of 35

376

selection to meet the newborn’s nutritional requirements, specific for each

377

species, and for their healthy development. To synthesize less protein but to

378

realize more functions through a heavier N-glycosylation may have been

379

advantageous for humans throughout evolution.

380

Fucosylation is one of the most important terminal decorations which endows

381

the N-glycans with important biological functions,(9, 28) and a higher percentage

382

of fucosylation in human milk N-glycans indicated a stronger biological

383

function. This is in agreement with the experimental results in this study.

384

Although the percentages of both sialylation and mannosylation in human milk

385

were similar to those in bovine milk, the absolute quantities of the two types of

386

N-glycans were higher in human milk. Therefore, the amounts of N-glycans

387

were in higher level in human milk and this can explain, to a certain extent, why

388

human milk N-glycans show a significantly higher anti-pathogenic activity than

389

bovine milk N-glycans. In addition, human milk contains a significantly higher

390

level of Neu5Ac, but in contrast to bovine milk, no Neu5Gc. The benefits of

391

Neu5Ac have been well described for humans, whereas Neu5Gc is not

392

endogenously synthesized in humans and believed to be immunogenic.(26)

393

Whether the richness in Neu5Ac and absence of Neu5Gc in human milk N-

394

glycome is partially associated with the higher anti-pathogenic activity of the

395

human milk N-glycome needs further studies.(27) Although only some of the N-

396

glycan structures presenting in both human and bovine milk were quantified,

397

it can be speculated from the quantitative difference that, besides of the

398

structural differences, these quantitative differences of the same structures are 18


Page 19 of 35


399

also responsible for the functional difference between human and bovine milk

400

samples.

401

The released N-glycans and the native proteome samples from human milk

402

exhibited significantly higher growth inhibitory effect against L. monocytogenes

403

than the corresponding bovine milk samples at the logarithmic phase of

404

listerial growth. The significant difference at the logarithmic growth phase is

405

presumed to be attributed to both of the structural and quantitative differences

406

of N-glycans in the milk samples. As reported in our previous study, there are

407

significant differences in N-glycan structures and in growth inhibition of

408

pathogens between human and bovine milk when human and bovine N-glycan

409

samples were used at the same concentration, indicating the exceptional effect

410

of structures.(9) In this study, the N-glycan samples compared were isolated

411

from the same volumes of milk, which means the human milk N-glycans is in

412

higher amount than the bovine milk N-glycans and the higher level of more

413

active human milk N-glycans undoubtedly further contributes to the higher

414

activity of growth inhibition against L. monocytogenes. This difference

415

disappeared after 24 h of bacterial cultivation, which is consistent with the

416

results seen in 24 h growth-inhibitory incubations of L. monocytogenes using

417

the halo assay in a previous study.(9)

418

Although the N-glycosylation made the milk proteins anti-listerial, the

419

activities of intact glycoproteins are much lower when compared to the

420

released N-glycans, indicating that N-glycan conjugated to proteins have lower

421

anti-pathogenic activity. Despite of the anti-pathogenic activity of N-glycans 19



Page 20 of 35

422

being greatly hampered when linked to protein, it is well known that the human

423

gut is a massive reservoir for diverse microorganisms,

424

commensal gut bacteria have been reported to be able to secrete endo-N-

425

glycanase which can release N-glycans from glycoproteins.(33,

426

possible that the N-glycosylation of milk proteins in the gut not only functions

427

as a signaling molecule in antibody-antigen recognition,(4, 35) but also acts as the

428

strong barrier against bacterial invasion once released from the proteins by

429

endo-N-glycanases from commensal bacteria.

430

Moreover, all types of tested samples from human milk had stronger overall

431

anti-listerial activities compared to the bovine milk samples, which may be also

432

associated with the structural and quantitative differences of the N-glycans. It

433

provides novel indications for the functional advantage of human milk over

434

bovine milk and sheds a light on the function of glycan-based natural

435

components which can be used in different foods such as baby formula to make

436

it functionally closer to mother milk.

437

Differing from the existing references which were most about the anti-

438

adhesion activity of milk N-glycans, (18, 36-38) we are now showing that milk N-

439

glycans can also inhibit the listerial growth. Moreover, N-glycosylation was

440

shown to be pivotal for the anti-pathogenic function of milk proteins. So far,

441

functional studies on protein N-glycosylation have been focused mainly on

442

their role in health and disease, and very little is known about their functions

443

in foods. It is the first time to show in this study that N-glycosylation confers

444

the milk whey proteins significant anti-pathogenic activity towards L.

(31, 32)

and several

34)

It is thus

20


Page 21 of 35


445

monocytogenes. Given that protein N-glycosylation is not only part of milk but

446

also an integral part of other foodstuffs such as eggs and meats, the function of

447

these N-glycans should be also analyzed for their anti-pathogenic activity.

448 449

Abbreviations

450

2AB, 2-aminobenzamide; BHI, brain heart infusion broth; DP, de-glycosylated

451

whey proteome; Gal, galctose; hmLF, human milk lactoferrin; Neu5Ac, N-

452

acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; Neu5Prop, N-

453

propionyl neuraminic acid; NG, released N-glycome; NP, native whey

454

proteome; Sia, sialic acid; TCA, trichloroacetic acid; UHPLC, ultra-high

455

performance liquid chromatography ； PNGase, peptide-N4-(N-acetyl-β-

456

glucosaminyl) asparagine amidase; OPD, O-phenylenediamine.

457 458

Funding Sources

459

This work was financially supported by the National Natural Science

460

Foundation of China (NSFC) 31871754 & 31371739 (L.L), the National Key

461

R&D Program of China 2017YFD0400604 (J.V) and the Fundamental Research

462

Funds for the Central Universities Y201700558 (J.V)

463

Supporting Information Available: no supporting information.

464 465

References

21



Page 22 of 35

466

1. Armand, M.; Hamosh, M.; Mehta, N. R.; Angelus, P. A.; Philpott, J. R.; Henderson, T. R.; Dwyer,

467

N. K.; Lairon, D.; Hamosh, P. Effect of Human Milk or Formula on Gastric Function and Fat

468

Digestion in the Premature Infant. Pediatric Research. 1996, 40, 429.

469

2. Lönnerdal, B. Nutritional and physiologic significance of human milk proteins. The American

470 471

Journal of Clinical Nutrition. 2003, 77, 1537S-1543S. 3. Calder, P. C. Functional Roles of Fatty Acids and Their Effects on Human Health. Journal of

472 473

Parenteral and Enteral Nutrition. 2015, 39, 18S-32S. 4. O'Riordan, N.; Kane, M.; Joshi, L.; Hickey, R. M. Structural and functional characteristics of

474 475

bovine milk protein glycosylation. Glycobiology. 2014, 24, 220-236. 5. Liu, B.; Newburg, D. S. Human Milk Glycoproteins Protect Infants Against Human Pathogens.

476 477

Breastfeeding Medicine. 2013, 8, 354-362. 6. Newburg, D. S.; Ruiz-Palacios, G. M.; Morrow, A. L. Human milk glycans protect infants against

478 479 480

enteric pathogens. Annual Review of Nutrition. 2005, 25, 37-58. 7.

Bode, L. Recent Advances on Structure, Metabolism, and Function of Human Milk Oligosaccharides. The Journal of Nutrition. 2006, 136, 2127-2130.

481

8. Gnoth, M.; Rudloff, S.; Kunz, C.; Kinne, R. Investigations of the in vitro transport of human milk

482

oligosaccharides by a Caco-2 monolayer using a novel high performance liquid

483

chromatography-mass spectrometry technique. Journal of Biological Chemistry. 2001, 276,

484

34363-34370.

485

9. Wang, W. L.; Wang, W.; Du, Y. M.; Wu, H.; Yu, X. B.; Ye, K. P.; Li, C. B.; Jung, Y. S.; Qian, Y. J.;

486

Voglmeir, J. Comparison of Anti-Pathogenic Activities of the Human and Bovine Milk N -

487

Glycome: Fucosylation is a Key Factor. Food Chemistry. 2017, 235, 167.

488 489

10. Ohtsubo, K.; Marth, J. D. Glycosylation in Cellular Mechanisms of Health and Disease. Cell. 2006, 126, 855-867. 22


Page 23 of 35


490

11. Mishra, A. K.; Driessen, N. N.; Appelmelk, B. J.; Besra, G. S. Lipoarabinomannan and related

491

glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis physiology and

492

host–pathogen interaction. FEMS Microbiology Reviews. 2011, 35, 1126-1157.

493 494 495 496

12. Moran, A. P.; Gupta, A.; Joshi, L. Sweet-talk: role of host glycosylation in bacterial pathogenesis of the gastrointestinal tract. Gut. 2011, 60, 1412. 13. Nothaft, H.; Szymanski, C. M. Protein glycosylation in bacteria: sweeter than ever. Nature Reviews Microbiology. 2010, 8, 765.

497

14. Croci, D.; Cerliani, J.; Dalotto-Moreno, T.; Méndez-Huergo, S.; Mascanfroni, I.; Dergan-Dylon,

498

S.; Toscano, M.; Caramelo, J.; García-Vallejo, J.; Ouyang, J. Glycosylation-Dependent Lectin-

499

Receptor Interactions Preserve Angiogenesis in Anti-VEGF Refractory Tumors. Cell. 2014, 156,

500

744-758.

501 502 503 504 505 506

15. Actor, J. K.; Hwang, S. A.; Kruzel, M. L. Lactoferrin as a Natural Immune Modulator. Current Pharmaceutical Design. 2009, 15, 1956-1973. 16. Jenssen, H.; Hancock, R. E. W. Antimicrobial properties of lactoferrin. Biochimie. 2009, 91, 1929. 17. van der Strate, B. W. A.; Beljaars, L.; Molema, G.; Harmsen, M. C.; Meijer, D. K. F. Antiviral activities of lactoferrin. Antiviral Research. 2001, 52, 225-239.

507

18. Barboza, M.; Pinzon, J.; Wickramasinghe, S.; Froehlich, J. W.; Moeller, I.; Smilowitz, J. T.;

508

Ruhaak, L. R.; Huang, J.; Lönnerdal, B.; German, J. B.; Medrano, J. F.; Weimer, B. C.; Lebrilla, C.

509

B. Glycosylation of human milk lactoferrin exhibits dynamic changes during early lactation

510

enhancing its role in pathogenic bacteria-host interactions. Molecular & Cellular Proteomics.

511

2012, M111.015248.

512

19. Yu, H.; Zhong, Y.; Zhang, Z.; Liu, X.; Zhang, K.; Zhang, F.; Zhang, J.; Shu, J.; Ding, L.; Chen, W.;

513

Du, H.; Zhang, C.; Wang, X.; Li, Z. Characterization of proteins with Siaα2-3/6Gal-linked glycans 23



Page 24 of 35

514

from bovine milk and role of their glycans against influenza A virus. Food & Function. 2018, 9,

515

5198-5208.

516 517

20. Allerberger, F.; Wagner, M. Listeriosis: a resurgent foodborne infection. Clinical Microbiology and Infection. 2010, 16, 16-23.

518

21. Dhama, K.; Karthik, K.; Tiwari, R.; Shabbir, M. Z.; Barbuddhe, S.; Malik, S. V. S.; Singh, R. K.

519

Listeriosis in animals, its public health significance (food-borne zoonosis) and advances in

520

diagnosis and control: a comprehensive review. Veterinary Quarterly. 2015, 35, 211-235.

521

22. Jackson, K. A.; Iwamoto, M.; Swerdlow, D. Pregnancy-associated listeriosis. Epidemiology and

522

Infection. 2010, 138, 1503-1509.

523

23. Kirjavainen, P. V.; Apostolou, E.; Arvola, T.; Salminen, S. J.; Gibson, G. R.; Isolauri, E.

524

Characterizing the composition of intestinal microflora as a prospective treatment target in

525

infant allergic disease. FEMS Immunology & Medical Microbiology. 2001, 32, 1-7.

526

24. Marques, T. M.; Wall, R.; Ross, R. P.; Fitzgerald, G. F.; Ryan, C. A.; Stanton, C. Programming

527

infant gut microbiota: influence of dietary and environmental factors. Current Opinion in

528

Biotechnology. 2010, 21, 149-156.

529

25. Wang, T.; Hu, X.C.; Cai, Z.P.; Voglmeir J.; Liu L. Qualitative and quantitative analysis of

530

carbohydrate modification on glycoproteins from seeds of Ginkgo biloba. Journal of

531

Agricultural and Food Chemistry. 2017, 65, 7669-7679.

532

26. Cao, C.; Wang, W. J.; Huang, Y. Y.; Yao, H. L.; Conway, L. P.; Liu, L.; Voglmeir, J. Determination

533

of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice. Journal of

534

Visualized Experiments. 2017, 125, e56030.

535

27. Hedlund, M.; Padler-Karavani, V.; Varki, N. M.; Varki, A. Evidence for a Human-Specific

536

Mechanism for Diet and Antibody-Mediated Inflammation in Carcinoma Progression.

537

Proceedings of the National Academy of Sciences USA. 2008, 105, 18936–18941. 24


Page 25 of 35


538

28. Wang, W. L.; Du, Y. M.; Wang, W.; Conway, L. P.; Cai, Z. P.; Voglmeir, J.; Liu, L. Comparison of

539

the bifidogenic activity of human and bovine milk N-glycome. Journal of Functional Foods.

540

2017, 33, 40-51.

541 542 543 544

29. Bode, L. The functional biology of human milk oligosaccharides. Early Human Development. 2015, 91, 619-622. 30. Okerblom, J.; Varki, A. Biochemical, Cellular, Physiological, and Pathological Consequences of Human Loss of N-Glycolylneuraminic Acid. ChemBioChem. 2017, 18, 1155-1171.

545

31. Penders, J.; Thijs, C.; van den Brandt, P. A.; Kummeling, I.; Snijders, B.; Stelma, F.; Adams, H.;

546

van Ree, R.; Stobberingh, E. E. Gut microbiota composition and development of atopic

547

manifestations in infancy: the KOALA Birth Cohort Study. Gut. 2007, 56, 661.

548

32. Claesson, M. J.; Jeffery, I. B.; Conde, S.; Power, S. E.; O’Connor, E. M.; Cusack, S.; Harris, H. M.

549

B.; Coakley, M.; Lakshminarayanan, B.; O’Sullivan, O.; Fitzgerald, G. F.; Deane, J.; O’Connor,

550

M.; Harnedy, N.; O’Connor, K.; O’Mahony, D.; van Sinderen, D.; Wallace, M.; Brennan, L.;

551

Stanton, C.; Marchesi, J. R.; Fitzgerald, A. P.; Shanahan, F.; Hill, C.; Ross, R. P.; O’Toole, P. W.

552

Gut microbiota composition correlates with diet and health in the elderly. Nature. 2012, 488,

553

178.

554

33. Garrido, D.; Nwosu, C.; Ruiz-Moyano, S.; Aldredge, D.; German, J. B.; Lebrilla, C. B.; Mills, D. A.

555

Endo-β-N-acetylglucosaminidases from infant-gut associated bifidobacteria release complex

556

N-glycans from human milk glycoproteins. Molecular & Cellular Proteomics. 2012, 9, 775-785.

557

34. Karav, S.; Le Parc, A.; Leite Nobrega de Moura Bell, J. M.; Frese, S. A.; Kirmiz, N.; Block, D. E.;

558

Barile, D.; Mills, D. A. Oligosaccharides Released from Milk Glycoproteins Are Selective Growth

559

Substrates for Infant-Associated Bifidobacteria. Applied and Environmental Microbiology.

560

2016, 82, 3622.

25



561 562

Page 26 of 35

35. Field, C. J. The Immunological Components of Human Milk and Their Effect on Immune Development in Infants. The Journal of Nutrition. 2005, 135, 1-4.

563

36. Aniansson, G.; Andersson, B.; Lindstedt, R.; Svanborg, C. Anti-adhesive activity of human

564

casein against Streptococcus pneumoniae and Haemophilus influenzae. Microbial

565

Pathogenesis. 1990, 8, 315-323.

566

37. Coppa, G. V.; Zampini, L.; Galeazzi, T.; Facinelli, B.; Ferrante, L.; Capretti, R.; Orazio, G. Human

567

Milk Oligosaccharides Inhibit the Adhesion to Caco-2 Cells of Diarrheal Pathogens: Escherichia

568

coli, Vibrio cholerae, and Salmonella fyris. Pediatric Research. 2006, 59, 377.

569

38. Lars, B.; Clemens, K.; Marion, M. R.; Konstantin, M.; Werner, S.; Silvia, R. Inhibition of

570

monocyte, lymphocyte, and neutrophil adhesion to endothelial cells by human milk

571

oligosaccharides. Thrombosis & Haemostasis. 2004, 92, 1402-1410.

572 573 574 575 576 577 578 579 580 581 582 583 584 585 26


Page 27 of 35


586

Table 1 Identification of glycosylated protein and relative band intensity using SDS-

587

PAGE Protein identity

Human whey protein (%)

Protein identity

Bovine whey protein (%)

1

35.27

5

4.27

2

10.92

6

8.26

3

8.67

7

28.34

4

4.27

Total

59.14

Total

40.86

588 589 590 591 592 593 594 595 596 597 598 599 600 601 27



602

Page 28 of 35

Table 2 The quantity comparison of selected N-glycan structures commonly shared between human and bovine milk

603 Colostrum (nmol/100 µl) Abbreviation

N-glycan structure

human milk

bovine milk

1st month (nmol/100 µl) P< 0.05

human milk

bovine

0.50±0.28

0.80±0.14

milk

3rd month (nmol/100 µl) P< 0.05

human milk

Bovine

0.59±0.20

1.14±0.70

milk

6th month (nmol/100 µl)

P< 0.05

human milk

Bovine

0.44±0.12

0.30±0.04

milk

P< 0.05

A2

0.61±0.25

1.11±0.47

A3

19.67±4.41

2.44±0.86

√

12.47±0.68

0.83±0.18

√

10.19±0.94

0.76±0.21

√

8.88±0.83

0.57±0.07

√

M5

12.57±6.66

1.04±0.37

√

4.67±1.29

0.36±0.08

√

3.28±0.89

0.33±0.08

√

2.65±0.74

0.22±0.07

√

FA2G1

4.44±3.62

2.84±0.48

3.36±1.23

2.06±0.5

1.66±0.73

1.74±0.58

1.17±0.45

0.71±0.09

M6

5.34±1.15

7.06±1.46

2.99±0.46

4.74±0.70

√

2.24±0.27

2.81±1.32

1.95±0.22

2.48±0.32

√

FA3G1

12.45±2.68

10.48±2.49

6.97±1.07

4.32±0.78

√

5.23±0.62

2.47±0.66

√

4.54±0.52

1.48±0.56

√

A2G2

20.14±5.8

7.71±3.64

√

13.14±3.12

4.12±1.14

√

8.94±2.64

3.33±1.41

√

7.65±1.18

2.08±0.85

√

FA2G2

82.82±20.87

17.71±4.32

√

43.77±5.49

7.25±1.56

√

25.63±4.01

3.58±1.10

√

14.62±1.92

2.06±0.3

√

M7

11.12±3.64

5.09±1.32

√

6.95±1.92

1.83±0.49

√

3.68±0.48

1.89±0.34

√

3.67±0.62

1.53±0.10

√

604 605

28


Page 29 of 35


606 607 608

Figure 1 The contents of total proteins (A) and N-glycans (B) in human milk (white) and bovine

609

milk (gray) at different lactation stages.

610 611 612 613

29



Page 30 of 35

614 615 616

Figure 2 SDS-PAGE profiles of human and bovine milk whey protein. M: Marker, lane A: Human

617

whey protein, lane B: Human whey protein with PNGase F incubated for 48 h, lane C:

618

Bovine whey protein, lane D: Bovine whey protein with PNGase F incubated for 48 h.

619 620 621 622 623 624 625 626 627

30


Page 31 of 35


628 629 630 631

Figure 3 The alterations of fucosylated strucures (A), sialylated structures (B) and high mannose structures (C) of human milk and bovine milk during lactation. (Note: All the data are shown as mean ± SD, n=15. Statistical method is one-way. * means

632

statistically significant difference at P

N-Glycosylation Plays An Essential and Species-Specific Role in Anti

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