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Comparative analysis of whey N-glycoproteins in human colostrum and mature milk using quantitative glycoproteomics Xueyan Cao, Dahe Song, Mei Yang, Ning Yang, Qing Ye, Dongbing Tao, Biao Liu, Rina Wu, and Xiqing Yue J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04381 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 8, 2017

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Journal of Agricultural and Food Chemistry

Comparative analysis of whey N-glycoproteins in human colostrum and mature milk using quantitative glycoproteomics

Xueyan Cao1, Dahe Song1, Mei Yang1, Ning Yang1, Qing Ye1, Dongbing Tao1, Biao Liu2, Rina Wu1, Xiqing Yue1* 1

College of Food Science, Shenyang Agricultural University, No.120 Dongling Road,

Shenyang, Liaoning, 110161, P.R.China 2

Inner Mongolia Yili Industurial Group Company Limited, Hohhot, Inner Mongolia,

151100, P. R. China

*

Corresponding Author:

Xiqing

Yue,

Tel:

+86

24-88488277;

Fax:

+86

[email protected]

1

ACS Paragon Plus Environment

24-88488277;

Email:


1

ABSTRACT

2

Glycosylation is a ubiquitous post-translational protein modification that plays a

3

substantial role in various processes. However, whey glycoproteins in human milk

4

have not been completely profiled. Herein, we used quantitative glycoproteomics to

5

quantify whey N-glycosylation sites and their alteration in human milk during

6

lactation; 110 N-glycosylation sites on 63 proteins and 91 N-glycosylation sites on 53

7

proteins were quantified in colostrum and mature milk whey, respectively. Among

8

these, 68 glycosylation sites on 38 proteins were differentially expressed in human

9

colostrum and mature milk whey. These differentially expressed N-glycoproteins

10

were highly enriched in “localization”, “extracellular region part”, and “modified

11

amino acid binding” according to gene ontology annotation, and mainly involved in

12

complement and coagulation cascades pathway. These results shed light on the

13

glycosylation sites, composition and biological functions of whey N-glycoproteins in

14

human colostrum and mature milk, and provide substantial insight into the role of

15

protein glycosylation during infant development.

16 17

KEYWORDS:

18

Human milk, colostrum, mature milk, label-free quantitation, whey glycoproteins,

19

N-glycosylation sites.

20

2


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INTRODUCTION

22

Human milk is the ideal first food for the newborn and developing infants, not only

23

because of its essential nutrients, but also because of its active biological constituents.

24

It is well known that breast-milk consumption is related to the healthy development of

25

infants and low rate of several diseases, including diarrhea as well as acute respiratory

26

and urinary tract infections.1-3 Considering these functions, human milk is regarded as

27

a functional food.4 The short-term and long-term benefits of breast milk are probably

28

due to its bioactive constituents, particularly proteins. A variety of proteins in human

29

milk, such as immunoglobulins, lactoferrin, and α-lactalbumin, perform bioactive

30

functions, including protection against infections and assistance in the development of

31

the immune system.5-7 These bioactive proteins in human milk potentially represent a

32

biological adaptation to meet the requirement of infants. As such, a comprehensive

33

understanding of the characteristics and functions of human milk proteins is

34

indispensable. And the better understanding of the bioactive proteins will in turn

35

provide substantial information for the improvement of infant products and infant

36

functional foods.

37

Milk proteins are classified as caseins, whey proteins, and milk fat globule

38

membrane (MFGM) proteins. Bovine milk whey proteins, which are widely used in

39

the preparation of functional foods for infants, play an important role in breastfeeding.

40

However, whey proteins in human and bovine milk are not exactly the same. Whey

41

proteins in human milk comprise one of the major part of the total proteins, which is

42

not the case in bovine milk.8 For the better improvement of infant formula milk, 3



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comprehensive investigation of human milk whey proteins is crucial. Thus, a number

44

of studies have focused on human whey proteomes. In addition, the composition of

45

whey proteins differs between human and bovine milk. Both major and minor

46

proteins in human milk whey,9-12 and mammalian milk whey13-16 have been

47

investigated by traditional methods and proteomic approaches. However, despite these

48

comprehensive studies, post-translational modifications (PTMs) of whey proteins in

49

human milk have not been studied in detail. PTMs influence the properties and

50

functions of the modified proteins. Therefore, for a complete understanding of milk

51

proteins, proteomic studies of milk proteins are not sufficient, and there is an urgent

52

need to investigate PTMs of proteins in human milk.

53

The major PTMs in milk proteins are phosphorylation and glycosylation. Casein is

54

the

main

milk

protein

that

undergoes

phosphorylation.

55

phosphorylation, glycosylation is more complicated. It is estimated that over 50% of

56

proteins in the human body undergo glycosylation, which increases the complexity of

57

the structure and function of the protein.17 The attached carbohydrates provide

58

recognition and adhesion sites, and glycoproteins are involved in multiple processes,

59

including cell-cell recognition, communication, signal transduction, immune defense,

60

protein folding, and adhesion.18-20 Thus, glycosylation of proteins in human milk is a

61

vital research field. Studies on milk protein glycosylation are primarily based on

62

glycomics and glycoproteomics. In regard to glycomics, the composition and

63

alterations of N-glycans in whole milk or on individual milk proteins were revealed in

64

human and mammalian milk.21-25 On the other hand, in regard to glycoproteomics, 4


Compared

with

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65

studies have focused on individual proteins and glycoproteomes. Notably, the

66

glycosylation sites of a few proteins in human and bovine milk were investigated.26-28

67

In human milk, 63 N-glycosylation sites on 32 proteins were identified by hydrophilic

68

interaction liquid chromatography (HILIC) coupled with MS.29 In addition, changes

69

in glycoprotein expression during various stages of lactation were explored.30,31 Most

70

recently, large-scale identification of N-glycoproteomes were performed in MFGM

71

from humans and seven mammals, and in whey from dairy milk.32,33 Although some

72

researches has focused on milk glycoproteins, the large-scale identification and

73

quantification of whey N-glycosylation sites in human milk by glycoproteomics has

74

not well been elucidated.

75

Human colostrum is the first milk that is generated after childbirth, and mature milk

76

is formed after a transition phase of 14 days. Interestingly, the composition of milk

77

proteins differs between colostrum, transitional milk, and mature milk, which are

78

produced during the different phases of lactation. For example, lactadherin and

79

immunoglobulins are more abundant in human colostrum, while fatty acid synthases

80

and binding proteins are less abundant in human colostrum than in mature milk.34

81

These results indicate that milk provides different nutrients and bioactive substances

82

as infants grow, to meet their special nutritional requirements. The glycosylation of

83

proteins also varies during lactation31, and changes in the glycoproteome could shed

84

light on the role of glycosylation during the different phases of lactation, for example

85

colostrum and mature milk. Therefore, the elucidation of the similarities and

86

differences of whey glycoproteins in human colostrum and mature milk is essential. 5



87

Therefore, the present work was aimed at identifying and quantifying the

88

N-glycopsylation sites on proteins and their alteration in colostrum and mature milk.

89

In this study, we applied N-glycoproteomics in combination with lectin enrichment,

90

deglycosylation in H218O, and quantitative label-free LC-MS/MS to analyze the

91

differences in whey N-glycoproteome between human colostrum and mature milk.

92

This research will facilitate a comprehensive understanding of the composition and

93

biological functions of human milk whey N-glycoproteome. The dymamic

94

glycosylation during lactation potentially reflects the different requirements of the

95

infants. Thus, elucidation of the quantitative changes in glycosylation sites on

96

glycoproteins from two different phases of lactation may shed some light on the

97

relationship between glycosylation and infant requirements during development.

98 99 100

MATERIALS AND METHODS Preparation of Whey Fraction

101

Human milk samples were donated by first-born mothers (25 to 30 years old).

102

Colostrum samples were collected from 30 healthy mothers on days 0–5 postpartum.

103

Samples of mature milk were collected from 30 healthy mothers 15 days to 6 months

104

postpartum. Milk samples were immediately frozen at –80 °C and kept at this

105

temperature until analyzed. For the preparation of whey, the samples of colostrum and

106

mature milk were thawed on ice and pooled respectively to reduce individual

107

differences and genetic variations. The pooled milk samples were centrifuged at

108

10000 g at 4 °C for 15 min. The fat fraction was carefully removed, and the skim milk 6


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samples were centrifuged at 150000 g at 4 °C for 1 h to separate the whey and casein.

110

The pellet was discarded, and the supernatant containing whey proteins was collected.

111

The concentration of whey proteins was measured using the Bradford assay (Bio-Rad,

112

CA, USA). The collection of human colostrum and mature milk samples was

113

approved by Shenyang Agricultural University and the Chinese Human Research

114

Ethical Committee, and conducted in accord with the Declaration of Helsinki and the

115

Nuremberg Code. Informed consent was obtained from all participants.

116

Trypsin Digestion of Whey Proteins

117

Milk proteins were processed according to the “filter-aided sample preparation”

118

(FASP) method.35 Eight-hundred micrograms of whey protein from human colostrum

119

and mature milk was dissolved in a solution consisting of 0.1 M NH4HCO3, Tris-HCl

120

pH 8.0, and 8 M urea in a 10-kDa ultrafiltration tube (Millipore, Bedford, MA, USA).

121

Then, the samples were alkylated with 50 mM iodoacetamide for 30 min in the dark.

122

After centrifugation at 14000 g for 15 min, the samples were diluted with UA buffer

123

(8 M urea, 150 mM Tris-HCl, pH 8.0), and then washed with 25 mM NH4HCO3 for

124

three times and the proteins were incubated with trypsin (proteins:trypsin, 50:1, w/w)

125

for 18 h at 37 °C. Digestion was stopped by adding formic acid; the peptides were

126

desalted on a solid phase extraction (SPE) C18 cartridge (Waters, MA, USA).

127

Glycosylated Peptide Enrichment by Lectin and Deglycosylation

128

The N-glycosylated peptides of whey proteins were enriched by lectin. Briefly, the

129

peptides were transferred into 30-kDa ultrafiltration tubes, and mixed with 50 µL

130

lectin mixtures containing 90 µg concanavalin A, 90 µg wheat germ agglutinin, and 7



131

90 µg Ricinus communis agglutinin, oscillated at 600 rpm for 1 min, and then

132

incubated for 1 h at room temperature. After centrifuging at 14000 g for 30 min, the

133

eluent was discarded, and the glycosylated peptides bound to the lectin mixtures were

134

washed four times with 1× BB buffer (0.5 mM CaCl2, 0.5 mM MnCl2, 0.25 mM NaCl

135

in 20 mM Tris-HCl, pH 7.3). The N-glycans were removed from the glycosylated

136

peptides by peptidyl N-glycosidase F (PNGase F) in H218O. After incubation at 37 °C

137

for 3 h, the deglycosylated peptides were eluted and analyzed.

138

Liquid Chromatography-tandem Mass Spectrometry Analysis

139

The deglycosylated peptides were resuspended in 0.1% FA and analyzed by

140

LC-MS/MS using a Q-Exactive mass spectrometer coupled with an Easy nLC

141

(Thermo Fisher Scientific, MA, USA). The samples were loaded onto a 100 µm × 20

142

mm C18 (3 µm; Thermo Fisher Scientific, CA, USA) pre-column at a flow rate of 10

143

µL/min. Chromatographic separation was performed on a 75 µm × 10 cm C18 (3 µm;

144

Thermo Fisher Scientific, CA, USA) nanocolumn. The high performance liquid

145

chromatography gradient was as follows: 0–55% buffer B (0.1% FA and 95%

146

acetonitrile) versus buffer A (0.1% FA) over 90 min at a flow rate of 300 nL/min. A

147

Q-Exactive mass spectrometer was used for the analysis (120 min). MS data was

148

acquired in positive ion mode with a precursor ion range of 300–1800. The resolution

149

was set to 70000 at m/z 200. The automatic gain control target was 3E6, maximum

150

ion injection time was 20 ms, and dynamic exclusion was 25 s. The ten most intense

151

ions were acquired for MS2 scans by higher-energy collisional dissociation at a

152

normalized collision energy of 27 eV. The resolution was set to 17500 at m/z 200, and 8


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the maximum IT was 60 ms.

154

Data Analysis

155

The label-free quantitative analysis of N-glycosylated peptides was performed

156

using the MaxQuant software version 1.3.0.5 (Max Planck Institute of Biochemistry

157

in Martinsried, Germany) based on a previous study.36 Tandem mass spectra were

158

searched against the Uniprot_human database (156639 entries; version 2017_01). Six

159

LC-MS/MS raw files were obtained from three replicates of human colostrum and

160

mature milk whey samples. The enzyme cleavage was set as trypsin, and the

161

maximum missed cleavage sites were two. Other search parameters were as follows:

162

fix modification was set as carbamidomethylation on cysteine; variable modifications

163

were set as oxidation on methionine and deamidation (18O) in asparagine; peptide

164

mass tolerance was set to 10 ppm; false discovery rate (FDR) thresholds for protein,

165

peptide and modification site were set to 0.01. Criteria were the assignment of major

166

peaks, occurrence of uninterrupted y- or b-ion series of more than 4 amino acids,

167

preferred cleavages N-terminal to proline bonds, the possible presence of a2/b2 ion

168

pairs, the presence of immonium ions, and mass accuracy. Only Glycosylated peptides

169

with a localization probability of ≥0.75 were accepted.37 Glycosylated sites identified

170

in at least two of the three replicates were used for comparative analysis. The average

171

intensity of peptides was used to compare fold change.

172

Bioinformatics Analysis

173

Gene ontology (GO) annotation and Kyoto Encyclopedia of Gene and Genomes

174

(KEGG) pathway analysis of proteins were performed using DAVID Bioinformatics 9



175

Resources 6.8 (https://david.ncifcrf.gov/). The GO categories with a P-value < 0.05

176

were used to for analysis. The gene symbol was obtained, and the analysis of the

177

protein–protein interaction network was performed using the IntAct database and

178

Cytoscape software (version 3.5.0). The Venn diagram of the identified

179

N-glycosylation sites and glycoproteins were carried out using the program Venny

180

on-line (http://bioinfogp.cnb.csic.es/tools/venny/index.html).38

181 182

RESULTS

183

Identification and Quantification of Whey N-glycoproteome by LC-MS/MS

184

Using lectin enrichment and LC-MS/MS, 110 N-glycosylation sites on 63

185

glycoproteins and 91 N-glycosylation sites on 53 glycoproteins were identified and

186

quantified in human colostrum and mature milk whey respectively, with at least 2

187

replicates and uniqueness to one protein group with 0.01 FDR (Table S1). Human

188

colostrum shared 68 glycosylation sites and 43 glycoproteins with mature milk; 42

189

glycosylation sites and 20 glycoproteins, and 23 glycosylation sites and 10

190

glycoproteins were uniquely identified and quantified in human colostrum and mature

191

milk, respectively (Figure 1a and 1b).

192

The number of glycosylation sites per protein varied from one to eight in human

193

colostrum and mature milk. Of these glycoproteins, 38 glycoproteins (60.1%) from

194

human colostrum, and 33 (62.3%) from mature milk contained a single glycosylation

195

site. Only two glycoproteins from human colostrum and one glycoprotein from

196

mature milk contained more than five glycosylation sites (Figure 1c). Polymeric 10


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immunoglobulin receptor (P01833) from human colostrum and mature milk contained

198

six and eight glycosylation sites, respectively.

199

Differentially Expressed N-glycosylation Sites and Glycoproteins in Milk Whey

200

Among the 68 shared whey glycosylation sites, 18 glycosylation sites on 14

201

glycoproteins were upregulated, while 11 glycosylation sites on 10 glycoproteins were

202

downregulated with a fold change of over 2-fold (colostrum/mature milk) (Table S2).

203

The levels of N-glycosylation site at N-497 of lactoferrin (P02788), N-551 of

204

galectin-3-binding protein (Q08380), and N-243 of haptoglobin (H0Y300) were

205

higher in human colostrum, while the levels of N-glycosylation site at N-125 of

206

galectin-3-binding protein, N-161 of folate receptor alpha (P15328), and N-86 of

207

clusterin (P10909) were the lower in colostrum than that in mature milk.

208

In addition, the glycosylation sites that were quantified in at least two of the three

209

replicates in one milk sample, but not detected in the three replicates of another milk

210

samples were defined as exclusively expressed glycosylation sites. We found that 25

211

glycosylated sites on 19 glycoproteins and 11 glycosylated sites on 8 glycoproteins

212

were exclusively expressed in human colostrum and mature milk, respectively (Table

213

S3). To comprehensively determine the differences in whey N-glycoproteins in human

214

colostrum and mature milk, the shared glycosylation sites with fold changes of over

215

2-fold and exclusively expressed glycosylation sites were identified as differentially

216

expressed whey N-glycosylation sites. The exclusively expressed glycosylation sites

217

in human colostrum and mature milk were attributed to the groups of upregulated or

218

downregulated glycosylation sites. Thus, 68 glycosylation sites on 38 proteins were 11



219

differentially expressed in human colostrum and mature milk.

220

Gene Ontology Analysis of Differentially Expressed Whey N-glycoproteins

221

To comprehensively determine the functions of the differentially expressed whey

222

N-glycoproteins in human colostrum and mature milk, the glycoproteins were

223

classified according to their involvement in “biological processes”, “molecular

224

functions”, and “cellular components” according to the GO annotations (Figure 2).

225

The differentially expressed glycoproteins were highly enriched in localization and

226

immune system process. Others were involved in response to stimulus, biological

227

regulation, regulation of biological process, and biological adhesion. With regard to

228

the cellular components, most differentially expressed glycoproteins originated from

229

extracellular region part and extracellular region, while the other glycoproteins

230

originated from the extracellular matrix, membrane, and organelle. The molecular

231

functions of differentially expressed whey glycoproteins were modified amino acid

232

binding, antigen binding, sulfur compound binding, carbohydrate derivative binding,

233

receptor activity, and enzyme regulator activity.

234

KEGG Pathway Analysis of Differentially Expressed Whey N-glycoproteins

235

The differentially expressed whey N-glycoproteins in human colostrum and mature

236

milk were related to 33 KEGG pathways; the first ten pathways are shown in Figure 3.

237

Complement and coagulation cascades, lysosome, and phagosomes were the major

238

pathways; others included protein processing in the endoplasmic reticulum (ER),

239

tuberculosis, extracellular matrix (ECM)-receptor interaction, focal adhesion,

240

Staphylococcus aureus infections, PI3K-Akt signaling pathway, and MicroRNAs in 12


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241

cancer.

242

Protein–protein Interaction Network of Differentially

243

N-glycoproteins

Expressed Whey

244

A highly connected protein–protein interaction network of the differentially

245

expressed whey N-glycoproteins in human colostrum and mature milk was

246

established (Figure 4). Each node of the network represented a glycoprotein, and each

247

edge represented its interactions. The main characteristic of a node is the number of

248

interaction partner proteins, which is defined as the degree of interaction. The network

249

contained 60 edges and 44 nodes, including 17 differentially expressed glycoproteins,

250

and 27 proteins interacted with them. Among the 17 differentially expressed

251

glycoproteins, 11 had upregulated N-glycosylation sites, 3 had downregulated

252

N-glycosylation sites, and 3 had both uprugulated and downregulated N-glycosylation

253

sites. Hypoxia upregulated protein 1 (HYOU1) had the highest interaction degree with

254

eight neighbors, haptoglobin (HP) had seven neighbors, and all the other differentially

255

expressed glycoproteins had five or less than five neighbors.

256 257

DISCUSSION

258

The short-term and long-term benefits of breast milk to infants are probably due to

259

its bioactive constituents, particularly proteins. Recently, milk whey proteome

260

compositions, comparisons, and alterations in human and mammalian milk during

261

various

262

technologies.9,10,12-14,39 However, milk glycoproteomes, as an important subset of milk

stages

of

lactation

were

studied

using

13


advanced

proteomics


Page 14 of 37

263

proteomes, are not well understood. In recent years, the elucidation of

264

N-glycoproteome of complex biological samples becomes feasible with the advent of

265

the large-scale N-glycosylation site annotation workflow by glycoproteomics method.

266

Interesting,

267

N-glycoproteome through glycoproteomics method. Previously, 63 N-glycosylation

268

sites on 32 proteins in human milk were identified by HILIC-MS; MFGM

269

N-glycosylation

270

N-glycosylation proteomes from dairy animals were revealed.29,32,33 These studies

271

provided fundamentally insights into human milk whey N-glycoproteome. Given that

272

glycosylation plays important roles in the structure, stability and function of milk

273

proteins, profiling the composition of glycoproteins and elucidating the alteration of

274

glycoproteions during lactation are crucial for the study of protein glycosylation in

275

human milk. While the aforementioned studies only focused on mature milk

276

N-glycoproteomes, we profiled and compared the whey N-glycoproteomes in human

277

colostrum and mature milk. Using a highly efficient N-glyco-FASP-based enrichment

278

equipped with high resolution LC-MS/MS, a total of 110 N-glycosylation sites on 63

279

proteins, and 91 N-glycosylation sites on 53 proteins were identified and quantified in

280

human colostrum and mature milk whey, respectively. By a label-free quantitative

281

proteomic approach, not only the N-glycoproteins were profiled in colostrum and

282

mature milk, but also the quantitative changes in glycosylation sites on glycoproteins

283

from two different phases of lactation were elucidated. And these findings may shed

284

some light on the relationship between glycosylation and infant requirements during

there

are

few

proteomes

studies

from

focus

humans

on

and

milk

dairy

14


protein

animals,

glycosylation

and

whey

Page 15 of 37


285

development.

286

Previously, the expression or glycosylation alterations of several abundant whey

287

proteins were elucidated by Froehlich et al. including tenascin, bile salt-stimulated

288

lipase (BSSL), lactoferrin, IgA and xanthine dehydrogenase (XD). In our study,

289

besides XD, the other proteins were all identified as N-glycoproteins as well (Table

290

S1). Compared with previous research conducted by Froehlich et al., the work

291

presented here shifts the aims from monitoring the most abundant protein

292

glycosylation using background-subtracted integrals of coomassie or pro-Q stained

293

electrophoretic speaks to quantifying the N-glycosylation sites by glycoproteomic

294

methods. Alpha-lactalbumin (P00709), highly abundant in human milk, owned one

295

glycosylation site at N-90 in both colostrum and mature milk in our study. This

296

unusual glycosylation site had been found in human milk previously40, and another

297

canonical glycosylation at N-64 had been found in human milk recently.29 However,

298

only one glycosylation site on alpha-lactalbumin was found in our research. Among

299

the immunomodulating proteins identified in previous studies, Ig mu chain C region

300

(P01871), IgA alpha1-heavy chain (P01876), IgA alpha2-heavy chain (P01877), Ig

301

heavy constant gamma 2 (P01859), Ig J chain (P01591) and polymeric Ig receptor

302

(P01833) were all identified as N-glycoproteins in our research. Mannose receptor

303

(P22897) had been identified as N-glycoproteins with one glycosylation site at

304

N-1205 in human milk whey29, while Froechlich et al. found it as a potentially

305

N-glycosylated based on Swiss-prot database. Here, besides one glycosylation site

306

reported before, we observed another three glycosylation sites on mannose receptor in 15



307

both human colostrum and mature milk. Galetin-3-binding protein (Q08380) was

308

described as a non-potential N-glycosylated protein by Froehlich et al., however, a

309

total of six glycosylation sites were quantified in our research. Previous, two

310

glycosylation sites were identified in human milk whey26; four were identified in

311

human MFGM.32 In addition, besides abundant whey glycoproteins, other

312

N-glycoproteins that were not usually accessible to proteomic analysis were detected

313

in our research. These findings increased the number of the known N-glycosylation

314

sites in human milk whey.

315

Elucidating the alterations of glycoproteins during lactation are crucial for the study

316

of protein glycosylation, we compared the whey N-glycoproteome and the

317

differentially expressed N-glycoproteins were found out in colostrum and mature milk.

318

To gain insight into the biological functions of whey N-glycoproteins in human

319

colostrum and mature milk, the differentially expressed glycoproteins were classified

320

according to the GO annotations. In previous studies, whey proteins were reported to

321

participate mainly in biological regulation and immune responses.12 However, the

322

biological functions and molecular functions of human whey N-glycoproteins are still

323

unclear. Recently, major whey N-glycoproteins in dairy milk were reported to be

324

involved in response to stimulus.33 In the current study, the differentially expressed

325

whey N-glycoproteins in human colostrum and mature milk were found to be

326

enriched mainly in localization, extracellular region part and sulfur compound binding.

327

It is noteworthy that 16 differentially expressed N-glycoproteins with 36

328

glycosylation sites were related to immune system process, of which 9 glycoproteins 16


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329

contained upregulated sites, 3 contained downregulated sites, and 4 contained

330

upregulated and downregulated sites (Table 1). The differences among these

331

immune-related glycoproteins between colostrum and mature milk suggest different

332

protein glycosylation needs for infants during various stages of growth. These

333

changes in glycosylation site expression may due to the different roles glycoproteins

334

played in infant protection against pathogens.41 Among the immune-related

335

glycoproteins, the glycosylation site at N-497 of lactoferrin (P02788) in colostrum

336

was upregulated 142.37-fold compared with that of lactoferrin in mature milk.

337

Lactoferrin (LTF), which mainly exists in the milk whey fraction, is a glycosylated

338

protein.42 Previously, it was reported that LTF, which acts as a vital bioactive

339

constituent for the protection of infants, decreased during the first two weeks after

340

birth.43 In this study, we also found an increased abundance of LTF N-glycosylation

341

sites in human colostrum compared with that in mature milk. Bioactive glycan chains

342

linked with glycosylation sites may play a role in inhibiting LTF proteolysis, and

343

glycosylation of LTF plays a role in multiple biological processes, including

344

iron-binding and nonspecific immune defense against pathogens by stimulating the

345

complement pathway and phagocytosis.44 In addition, we found 5 upregulated and 3

346

downregulated glycosylation sites on polymeric immunoglobulin receptor (PIGR) in

347

human colostrum and mature milk. PIGR is an immunomodulating protein, which is

348

an important component of the immune system. Previous studies reported several

349

glycosylation sites on PIGR;29,33 however, herein, we report alterations in its

350

glycosylation sites during lactation. The GO analysis of N-glycoproteome, which is a 17



351

complementary subclass of proteome, revealed the biological functions of whey

352

N-glycoproteins and their differences in human colostrum and mature milk, which

353

contributes to a complete understanding of the functions of whey proteins.

354

Moreover, the differentially expressed N-glycoproteins were mainly involved in

355

complement and coagulation cascades according to our KEGG pathway analysis

356

(Figure S1). Four glycoproteins associated with complement and coagulation cascades

357

including C4-A (C4A), clusterin (CLU), CD59 glycoprotein (CD59), and

358

plasminogen (PLG) were differentially expressed in human colostrum and mature

359

milk (Fig. 5). The glycosylation sites at N-226 of C4A (P0C0L4), N-43 of CD59

360

(P13987), and N-308 of PLG (P00747) were upregulated, while that at N-86 of CLU

361

(P10909) was downregulated in colostrum milk compared with mature milk. The

362

complement system, which acts as a mediator of the innate immune system and a

363

nonspecific defense against pathogens, directly affects cell lysis or induces

364

inflammatory responses.45 Components of the complement system include a series of

365

inactive zymogens, and some of them are glycosylated. These complement

366

components can be activated by proteolysis, and induce cascade reactions, and

367

ultimately, the membrane attack complex (MAC), which can induce cell lysis, is

368

formed.46 PLG is an independent natural C3 and C5 convertase, which activates C3,

369

C5, and the other components of the cascade.47 The biological activity of the

370

complement pathway does not just depend on cell lysis; anaphylatoxins including C4a,

371

C3a, and C5a in the complement pathway have some inflammatory effects that can

372

activate and degranulate mast cells and platelets.48-50 Since the complement system 18


Page 18 of 37

Page 19 of 37


373

may also damage host tissues, it must be regulated. CLU and CD 59 are major

374

regulators of the complement pathway, and they can prevent MAC formation, and

375

thus, inhibit complement-mediated cytolysis.51,52 These proteins involved in the

376

complement and coagulation pathways were also reported in bovine milk, human milk

377

and plasma.53-55 Herein, we reveal differences in their glycosylation during lactation,

378

which may provide insight into these complement proteins from a modification

379

perspective, and shed light on the functions of glycosylated proteins in human milk.

380

Furthermore, the protein–protein interaction network of the differentially expressed

381

whey N-glycoproteins was delineated in the current study. Protein–protein

382

interactions, which modulate and mediate protein functions, are reported to play a

383

crucial role in various biological processes. Previously, the protein–protein interaction

384

pathways of milk proteins have been reported by D’Alessandro et al.56 Here, we

385

mainly focus on the interactions between the differentially expressed N-glycoproteins

386

in human colostrum and mature milk. Of the 17 differentially expressed proteins in

387

the interaction network, PIGR was differentially abundant with 8 glycosylation sites

388

(5 upregulated and 3 downregulated), HP with five glycosylation sites (2 upregulated

389

and 3 downregulated), and the other proteins with less than five glycosylation sites.

390

These differences in glycosylation level may be responsible for the functional

391

differences between human colostrum and mature milk for infants. According to the

392

GO annotations, the differentially expressed glycoproteins in the network were

393

mainly involved in localization, immune system processes, and responses to stimuli.

394

Interestingly, we identified some N-glycoproteins with high degrees of interactions, 19



395

including HYOU1, HP, galectin-3-binding protein (LGALS3BP), CD59, and CLU.

396

These glycoproteins interacted with each other via one or two proteins, and it is

397

thought that a change in one protein might affect the other, thus, glycoproteins with

398

high degrees of interaction may be the crucial factors which could influence a variety

399

of biological processes. Our study provides some novel insights into the protein–

400

protein interactions of differentially expressed whey N-glycoproteins in human

401

colostrum and mature milk; however, the specific changes caused by these protein–

402

protein interactions in milk require further investigation.

403

In conclusion, for the first time, we have revealed quantitative differences in the

404

whey N-glycosylation sites in human colostrum and mature milk. The differentially

405

expressed glycoproteins in human milk may reflect changes in nutritional

406

requirements of infants during different stages of growth. Our results will contribute

407

to a better understanding of the biological functions, especially immune functions,

408

and complex interactions of N-glycoproteins in human colostrum and mature milk,

409

unveil the needs of infants during lactation, and may lead to improvements in infant

410

formula compositions.

411 412

ASSOCIATED CONTENT

413

Supporting Information

414

Table S1. Glycosylation Sites and Glycoproteins Identified and Quantified in

415

Human Colostrum and Mature Milk Whey. Table S2. Glycosylation Sites Shared in

416

Human Colostrum and Mature Milk Whey. Table S3. Table S3. Glycosylation Sites 20


Page 20 of 37

Page 21 of 37


417

Exclusively Expressed in Human Colostrum and Mature Milk Whey. Figure S1.

418

Differentially expressed whey N-glycoproteins involved in complement and

419

coagulation cascades.

420 421

AUTHOR INFORMATION

422

Corresponding Author

423

Xiqing Yue, Email: [email protected];

424

Funding Sources This work was supported by the “Twelfth Five Year” National Science and

425 426

Technology Plan Project, Grant (2013BAD18B03-02).

427 428

ABBREVIATIONS USED

429

MFGM, milk fat globule membrane; PTM, post-translational modification; MS,

430

mass spectrometer; FASP, filter-aided sample preparation; FA, formic acid; SPE, solid

431

phase extraction; HPLC-MS/MS, high-performance liquid chromatography/tandem

432

mass spectrometry; PNGase F, peptidyl N-glycosidase F; FDR, false discovery rate;

433

GO, Gene ontology; KEGG, Kyoto Encyclopedia of Gene and Genomes; ER,

434

endoplasmic reticulum; ECM, extracellular matrix; HC, human colostrum; HM,

435

human mature milk.

436 437

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438

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29



Page 30 of 37

FIGURE CAPTIONS

Figure 1. Characterization of whey N-glycoproteomes. (a) Comparison of protein N-glycosylation sites in human colostrum (HC) and mature milk (HM). (b) Comparison of N-glycoproteins in HC and HM. (c) Number of N-glycosylation sites per protein in HC and HM whey.

Figure 2. GO annotation of differentially expressed whey N-glycoproteins in human colostrum and mature milk.

Figure

3.

KEGG

pathway

analysis

of

differentially

expressed

whey

N-glycoproteins in human colostrum and mature milk. The first ten pathways are shown. ER, endoplasmic reticulum. ECM, extracellular matrix.

Figure 4. Protein–protein interaction network of differentially expressed whey N-glycoproteins in human colostrum and mature milk. Disconnected nodes are hidden.

30


Page 31 of 37


Table 1. The Differentially Expressed Whey N-glycoproteins in Immune System Process. Human colostrum (HC); human mature milk (HM). No

UniProt

.

accession

1

Q6UX06

Sites Description

Changes

HC/HM

(N-) Olfactomedin 4

136

↑

6.00

253

↑

HC

193

↑

HC

1067

↑

2.19

↑

HC

209

↑

HC

46

↑

HC

440

↑

3.10

2

P07996

Thrombospondin 1

3

P11279

Lysosome-associated membrane 103 glycoprotein 1

4

P01871

Ig mu chain C region

5

P25311

Zinc-alpha-2-glycoprotein

128

↑

HC

6

P02788

Lactoferrin

534

↑

HC

497

↑

142.37

156

↑

3.92

7

P13987

CD59 glycoprotein

43

↑

2.26

8

H0Y300

Haptoglobin

277

↓

0.45

243

↑

42.50

220

↑

HM

229

↑

HM

31



9

O00300

Page 32 of 37

247

↓

HC

Tumor necrosis factor receptor 289

↑

HM

↓

0.48

immunoglobulin 469

↑

2.25

499

↑

2.04

500

↑

2.04

421

↑

HC

429

↑

HC

90

↓

HM

83

↓

HM

96

↓

HM

superfamily member 11B 10

P01591

Immunoglobulin J chain

11

P01833

Polymeric receptor

71

12

P0C0L4

Complement C4-A

226

↑

15.67

13

P10909

Clusterin

86

↓

0.12

14

P01876

Immunoglobulin heavy constant 493

↑

6.63

15

Q08380

alpha1

125

↓

0.02

Galectin-3-binding protein

551

↑

105.38

398

↑

HC

1160

↑

4.07

1205

↓

0.38

16

P22897 Mannose receptor 1

32


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33



34


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35



36


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GRAPHIC FOR TABLE OF CONTENTS

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