Comparative Site-Specific N-Glycosylation Analysis of

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Comparative site-specific N-glycosylation analysis of lactoperoxidase from buffalo and goat milk using RP-UHPLC-MS/MS reveals distinct glycan pattern Gnanesh Kumar B S, Prasad Mohan Reddy, and Sanjay Kottekad J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03243 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

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Journal of Agricultural and Food Chemistry

1

Comparative site-specific N-glycosylation analysis of lactoperoxidase from buffalo and goat

2

milk using RP-UHPLC-MS/MS reveals distinct glycan pattern

3

Gnanesh Kumar B S1*, Prasad Mohan Reddy2 and Sanjay Kottekad1.

4

1Department

Mysuru- 570020, Karnataka, India.

5 6

of Biochemistry, CSIR-Central Food Technological Research Institute (CFTRI),

2St.

Joseph’s College (Autonomous), Shanthinagar, Bengaluru- 560027, Karnataka, India.

7

* Correspondence for Gnanesh Kumar B S: Telephone: +918212514876;

8

email: [email protected], [email protected]

9 10 11 12 13 14 15 16 17 18 19 1 ACS Paragon Plus Environment


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ABSTRACT

22

N-glycan pattern of lactoperoxidase (LPO) from buffalo and goat milk was analysed with

23

corresponding site of attachment. The enzyme was purified from whey on cation exchange

24

chromatography, proteolyzed using chymotrypsin and resulting (glyco)peptides were directly

25

analysed on reverse phase ultra-high performance liquid chromatography coupled to ESI-Q-TOF

26

MS in tandem mode. N-glycans such as high mannose, complex and hybrid types were identified

27

in buffalo and goat LPO. Among sialylated complex and hybrid types, the terminal Neu5Ac

28

linked to either LacNAc/LacdiNAc found exclusively in buffalo whereas Neu5Gc linked to

29

LacdiNAc was predominant in goat LPO. N-glycans at Asn6 and Asn349 in buffalo LPO were

30

completely core fucosylated while these sites in goat LPO showed differential fucosylation.

31

Differential occupancy was observed at Asn112 with or without non-fucosylated complex and

32

hybrid types, whereas mainly high mannose glycans was found in Asn222 in both the LPO. The

33

presence of glycan isomers in buffalo and goat LPO was observed. Despite the presence of

34

distinct complex and hybrid glycans the common glycosylation features in buffalo and goat LPO

35

was identified, and is comparable with bovine LPO. The finding could be useful in exploring

36

beneficial role of these glycans as functional ingredients for food products.

37 38

KEYWORDS

39

Lactoperoxidase, mammals, N-glycans, site-specific, tandem mass spectrometry,

40

2 ACS Paragon Plus Environment

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INTRODUCTION

44

Milk is the primary source of nutrition for all mammalian infant that comprises various

45

components such as free oligosaccharides, proteins, peptides, lipids, and micronutrients. These

46

components, other than providing nutrition; also protects the neonate from harmful

47

microorganisms1. In addition to free oligosaccharides, there are bound glycans in the form of

48

glycoproteins and glycolipids that contain covalently attached oligosaccharides. Milk

49

glycoproteins are broadly classified into casein, whey glycoproteins and milk fat globule

50

membrane proteins (MFGM). Majority of the glycolipids are associated with the MFGM2. The

51

whey glycoproteins such as lactoferrin, immunoglobulins, lactoperoxidase predominantly

52

possess N-glycans2. In eukaryotes, N-glycans are linked to Asn in a consensus sequence Asn-X-

53

Thr/Ser/Cys, and are structurally diverse, categorized in to high mannose, complex and hybrid

54

type. The composition and structure of N-glycans at each site vary considerably depending on

55

the cellular conditions. N-glycans can influence conformation, secretion, binding of protein, thus

56

involving in many biological processes3, 4.

57

Lactoperoxidase, a member of heme peroxidase serves as antimicrobial agent in mammalian

58

milk. It catalyses the conversion of thiocyanate to hypo thiocyanate- an antimicrobial

59

component, by reducing hydrogen peroxide to water5. Thus, LPO serves as natural defence

60

component in milk and along with hydrogen peroxide and thiocyanate forms Lactoperoxidase

61

system (LPS)6. LPO has found variety of applications in food, dairy and other sectors as an

62

antimicrobial agent. The activation of LPS has shown the extension of shelf life of milk as well



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as component in cosmetics and oral hygiene7-9. Bovine, buffalo and goat milk contains higher

64

concentrations of LPO than human10. Mature LPO is a single polypeptide chain consisting of 612

65

amino acids in bovine and is predicted for buffalo and goat, while in human it is 632 residues

66

long and exhibit high degree of sequence and structural homology. LPO from bovine, buffalo

67

and goat possesses five potential N-glycosylation sites at Asn6, Asn112, Asn222, Asn258 and Asn349

68

consensus tripeptide. Human LPO comprises four sites: Asn26, Asn132, Asn242, and Asn278 with

69

same sequence motif as seen in bovine and other11, 12 (Figure S1).

70

The site-specific glycosylation analysis of bovine LPO indicated the occupancy of five sites,

71

with glycans composition being predicted as high mannose, complex, hybrid types13.

72

Crystallographic analysis of LPO from buffalo and goat milk has also shown the occupancy of

73

sites at Asn112, Asn222, Asn258 and Asn449 14, 15, however, the information on structure or sequence

74

of glycans on these sites in the mammalian lactoperoxidase studied so far remains elusive.

75

Similar to free oligosaccharides, bound glycans in milk can also exert bio functionalities such as

76

promoting probiotics growth, preventing the adhesion of pathogens16,

77

exhibits isomerism having more or less similar monosaccharides composition but differ in

78

arrangements18. To understand these properties it is essential to characterize them. Here in we

79

determined the site specific N-glycans of buffalo and goat LPO with simple digestion protocol.

80

The purified LPO was subjected to chymotrypsin digestion and without any further processing of

81

glycopeptides directly applied on reverse phase liquid chromatography and analysed on Q-TOF

82

MS. The tandem MS experiments resulted in the identification of N-glycans composition and

83

sequence with corresponding site of attachment in both the LPO.

84

MATERIALS AND METHODS

85

Materials. Raw milk from buffalo was obtained from the local farm while goat milk was 4 ACS Paragon Plus Environment

17.

Bound glycans also

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procured from Yashodavana goat farm, Mysuru. Ammonium bicarbonate, CM-Sephadex C50

87

ion exchange matrix, dithiothreitol (DTT), dialysis tubing, iodoacetamide, proteomics grade

88

chymotrypsin, Trizma Base were procured from Sigma (MO, USA). Vivaspin 20 ultrafiltration

89

units were obtained from Sartorius Stedim Biotech (Germany). Dual stain precision plus protein

90

marker and micro biospin columns were procured from Bio-Rad (CA, USA). LC-MS grade

91

acetonitrile, formic acid and water were procured from J.T. Bakers (PA, USA). All other

92

chemicals used in this study were of high purity and obtained from local vendors.

93

Preparation of whey and purification of enzyme. 200 mL each of Buffalo and goat milk in

94

two batches was used for whey preparation according to protocol described earlier19. The

95

lactoperoxidase activity in the whey was assayed using guaiacol and hydrogen peroxide as

96

substrate19. The whey was brought to 50 % ammonium sulfate saturation by adding solid

97

ammonium sulfate and stirred overnight at 4 oC. After centrifugation at 10000 RPM for 20 mins,

98

the clear supernatant was subjected to 80% saturation to obtain enzyme enriched fraction20. The

99

pellet was dialysed extensively against Tris HCl, 50 mM pH 8.0 (column buffer) and was applied

100

on CM-Sephadex C-50 cation exchange matrix equilibrated with the same buffer. The matrix

101

was washed with column buffer to remove the unbound proteins until the OD at A280 nm was
700 having intensity of >500 cps.

129

Data analysis. The MS data files were processed using PeakView 2.1. The mascot generic

130

format (.mgf) was created and used for online Mascot MS/MS Ions search (Matrix science, UK). 6 ACS Paragon Plus Environment

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The search was performed with following parameters: database, SwissProt (2018/04); taxonomy,

132

other mammalia; enzyme, chymotrypsin (FLWY) with three missed cleavages; with default mass

133

error. The tandem mass spectra (MS/MS) were screened manually for the presence of commonly

134

found sugar oxonium ions such as m/z 204.09 (HexNAc + H)+1, m/z 366.14 (HexNAc + Hex

135

+H)+1 and m/z 528.20 (HexNAc+2Hex)+1 that represents glycopeptide fragmentation. The glycan

136

sequence was determined by manual evaluation of B- and Y- type ions as reported previously21,

137

22.

138

proximal HexNAc residue. The mass of peptide portion was used to determine the amino acid

139

sequence by comparing with the theoretical mass of peptides generated by in-silico chymotrypsin

140

digestion of the LPO using MS-Digest (prospector.ucsf.edu). The theoretical mass of

141

glycopeptides was determined using MS-Product online tool (prospector.ucsf.edu). The

142

monosaccharides are represented in the figures are according to consortium for functional

143

glycomics.

144

RESULT AND DISCUSSION

145

Glycans exhibit microheterogeneity at each site and through MS analysis, information on the

146

glycan types can be obtained by evaluating the set of precursor ions having mass differences that

147

corresponds to certain sugar moieties13. However, by tandem MS analysis (MS/MS) the

148

composition and sequence of glycans can be determined23. MS/MS of multiply charged N-

149

glycopeptides mainly results in B- and Y-ions arising from glycans and to some extent from

150

peptide backbone which will allow to deduce glycan sequence and assigning them to specific

151

site24. Further the MS analysis of whole protein digest will be helpful in determining the variable

152

occupancy of glycosylation sites (macroheterogeneity)4.

The intact peptide mass was determined using Y1 ions that corresponds to peptide with



153

The Mascot search revealed that the purified protein is indeed lactoperoxidase and several

154

peptides were identified matching to both bovine and buffalo LPO due to high sequence

155

homology (Figure S3A and S3B). In the tandem spectra of N-glycopeptides derived from buffalo

156

LPO, we observed the occurrence of unique diagnostic ions at m/z 292.11 [Neu5Ac +H]+1, m/z

157

495.19 [Neu5Ac + HexNAc + H]+1, m/z 657.25 [Neu5Ac + Hex + HexNAc + H]+1, m/z 698.27

158

[Neu5Ac+2HexNAc+ H]+1 indicate the presence of N-acetylneuraminic acid. But goat LPO

159

spectra predominantly comprised ions at m/z 308.10 [Neu5Gc + H]+1 511.18 [Neu5Gc +

160

HexNAc + H]+1, 714.27 [Neu5Gc + 2HexNAc + H]+1 matching to N-glycoylneuraminic acid. A

161

study by Watanabe et al25 has showed the presence of sialylated glycans in bovine LPO, however

162

mass spectrometry analysis could not confirm it13. We observed Neu5Ac exclusively in buffalo

163

and Neu5Gc in goat LPO suggesting the presence of sialylated glycans in species specific

164

manner. Similarly goat milk lactoferrin glycans has shown to comprise predominantly Neu5Gc26.

165

Figure 1(A) represents the MS/MS of N-glycopeptide precursor ions derived from buffalo LPO

166

at m/z 1229.9+3 corresponding to the sialylated hybrid glycans. The presence of ions at m/z

167

292.11 [Neu5Ac + H]+1 and 274.10 [Neu5Ac – H2O + H]+1 unambiguously represent the

168

terminal N-aceyl neuraminic acid and additionally the ions at m/z 407.17 [2HexNAc + H]+1

169

indicate the presence of LacdiNAc (GalNAcGlcNAc). Neu5Ac found attached to this moiety as

170

evidenced from the presence of ions at m/z 495.19 [Neu5Ac + HexNAc+H]+1 and 698.27

171

[Neu5Ac + 2HexNAc + H]+1. The evaluation of other B- and Y- ions revealed the core

172

fucosylation with glycan composition NeuAc1HexNAc2Hex4HexNAc2(dHex) corresponding to

173

sequence NeuAc1GalNAc1GlcNAc1Man4GlcNAc2(Fuc). The Y0 was observed at 1790.96+1 (Y1

174

at m/z 1993.03+1) matched to peptide mass DTTLTN6VTDPSLDLTAL1-17 which is N-terminus

175

of LPO. Further confirmation for this peptide was obtained by the presence of fragment ions at


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m/z 829.49+1 for peptide sequence PSLDLTAL. Complex type glycans were also observed at

177

Asn6 as represented in Figure 1B (m/z 1200.56+3) which is non-sialylated but fucosylated with

178

structure having both terminal LacNAc [GalGlcNAc] and LacdiNAc [GalNAcGlcNAc] moieties.

179

In total, 6 different fucosylated glycan structures were observed on Asn6 with almost equal

180

abundance of complex and hybrid types (Table 1).

181

MS/MS of N-glycopeptides at m/z 1187.55+3 derived from goat LPO having the peptide

182

backbone with Asn6 (Y1 at m/z 1993.03+1) revealed the presence of hybrid glycans (Figure 1C).

183

The ions at m/z 407.17 [2HexNAc + H]+1 indicate the presence of LacdiNAc (GalNAcGlcNAc)

184

and

185

HexNAc2Hex5HexNAc2(dHex) with core fucosylation that corresponds to glycan sequence

186

GalNAc1GlcNAc1Man5GlcNAc2(Fuc). The glycans at this site also harbours sialylated hybrid or

187

complex glycans with Neu5Gc. As represented in Figure 1D (m/z 1235.6+3), ions at m/z 308.10

188

and 290.09 indicated the presence of Neu5Gc and Neu5Gc-H2O respectively and the attachment

189

exclusively to LacdiNAc was confirmed by the ions at m/z 714.27 [Neu5GcGalNAcGlcNAc +

190

H]+1

191

corresponding to hybrid type. Interestingly, the spectrum also comprised ions at m/z 495.19

192

[Neu5Ac + HexNAc + H]+1 and m/z 698.27 [Neu5Ac + 2HexNAc + H]+1 similar to buffalo LPO

193

(Figure

194

NeuAc1GalNAc1GlcNAc1Man5GlcNAc2. The peptide backbone was confirmed by the presence

195

of intense fragment ions at 829.48+1. Altogether, goat LPO showed 8 different glycan structures

196

at Asn6 having ~90 % hybrid and ~10 % complex type (Table 1). In contrast to previous

197

structural study on buffalo and goat LPO where N-terminus was identified as

198

SWEVGCGAPVPLVK14, 15, we obtained the N-terminal peptide DTTLTNVTDPSLDLTAL1-17

the

complete

resulting

1A)

in

evaluation

the

indicating

of

glycan

the

the

spectrum

sequence

presence

revealed

the

glycan

composition

NeuGc1GalNAc1GlcNAc1Man4GlcNAc2(Fuc)

of

Neu5Ac


with

glycan

sequence


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199

in the glycosylated form, which is in accordance with bovine LPO13. N-glycans at Asn6 in bovine

200

LPO found to be only neutral complex type with or without fucose13, but in buffalo LPO we

201

observed the presence of both sialylated/neutral fucosylated complex and hybrid glycans while

202

goat LPO showed predominantly sialylated/neutral hybrid glycans with differential fucosylation.

203

MS/MS analysis of N-glycopeptides derived from buffalo LPO at m/z 1194.87+3 revealed

204

sialylated complex N-glycans without fucosylation (Figure 2A). The glycan composition was

205

found

206

NeuAcGalNAc2GlcNAc2Man3GlcNAc2 with LacdiNAc residues. The peptide backbone (Y0)

207

mass at m/z 1586.80+1 (Y1 at m/z 1789.93+1) matches to sequence LDEDGVLDQN112RSLL103-116,

208

showing substitution for Glu106 to Asp106. Glycans with sialyl residue linked to LacNAc

209

(GalGlcNAc)

210

NeuAcGal2GlcNAc2Man3GlcNAc2 (m/z 1167.86+3)(Figure 2B).

211

structures including high mannose, hybrid types were found to occupy Asn112 with higher

212

abundance of complex glycans (> 75%) (Table 1). N-glycopeptides with peptide backbone

213

AREVSNKIVGYLDEDGVLDQN112RSLL were also generated due to missed cleavage of

214

chymotrypsin and the glycoforms/isomers at this site is summarised in Table S1. The MS/MS of

215

N-glycopeptides at m/z 1187.81+4 derived from goat LPO revealed sialylated hybrid glycans

216

having

217

NeuGc1GalNAc1GlcNAc1Man5GlcNAc2 without fucosylation. The Y0 ions at m/z 1409.24+2 (Y1

218

at m/z 1511.32+2) matched to peptide backbone AREVSNKIVGYLDEEGVLDQN112RSLL

219

(Figure 2C). Sialylated but non-fucosylated complex type glycans with Neu5Gc (m/z 1208.83+4)

220

were also observed at this site (Figure 2D). Six different glycans mainly complex (69 %) and

221

hybrid (31%) types were found at Asn112 (Table 1). N-glycopeptides with peptide backbone

to

be

NeuAc1HexNAc4Hex3HexNAc2

were

composition

also

present

in

corresponding

the

same

NeuGc1HexNAc2Hex5HexNAc2


to

site

glycan

with

sequence

sequence

Overall 8 different glycans

corresponds

to

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222

LDEEGVLDQN112RSLL were also produced for goat LPO (Table S1). In bovine LPO, Asn112

223

showed 85 % glycosylation with mainly non-fucosylated complex glycans13. The Mascot search

224

revealed the presence of peptide precursor ions at m/z 935.50+3 in buffalo and m/z 939.84+3 in

225

goat LPO corresponding to peptide sequence AREVSNKIVGYLDEDGVLDQN112RSLL and

226

AREVSNKIVGYLDEEGVLDQN112RSLL, respectively, indicating the absence of glycans at

227

Asn112 (Figure S3A and S3B). Based on the relative abundance of glycosylated and non-

228

glycosylated forms Asn112 found to undergo 65 % and 40 % glycosylation in buffalo and goat

229

LPO respectively. Thus the Asn112 exhibited variable occupancy of glycans known as macro-

230

heterogeneity4, and site-specific micro heterogeneity revealed the presence of mainly non-

231

fucosylated complex or hybrid types which is suggestive of similar processing of Asn112 site in

232

bovine, buffalo and goat.

233

The MS/MS of precursor ions at m/z 1164.02+2 derived from both buffalo and goat LPO revealed

234

sequential loss of 162 u corresponding to hexose i.e. mannose (Figure 3). In buffalo, glycans

235

comprising Man4-8 whereas in goat Man4-9 was identified (Table 1). The Y0 ion at m/z 786.46+1

236

(Y1 at m/z 989.54+1) matched to peptide sequence RN222LSSPL. The presence of high mannose at

237

Asn222 is in agreement with bovine LPO harbouring Man5-913. The presence of Man4GlcNAc2 (8-

238

9%) is distinctive in a site that predominately harbours high mannose type and similar structures

239

are also reported in bovine and goat lactoferrin26, 27.

240

The fragmentation pattern of N-glycopeptides at m/z 1141.17+3 derived from buffalo LPO

241

revealed

242

NeuAc1Gal2GlcNAc2Man3GlcNAc2(Fuc) (Figure 4A). In addition to peaks at m/z 366.14

243

[GalGlcNAc/ManGlcNAc],

244

[GalNAcGlcNAc], 495.19 [Neu5AcGalNAc] and 698.27 [Neu5AcGalNAcGlcNAc] indicate the

sialylated

and

m/z

fucosylated

657.25

complex

glycan

[Neu5AcGalGlcNAc],


ions

with

at

sequence

m/z

407.17


245

presence of hybrid glycans NeuAc1GalNAc1GlcNAc1Man5GlcNAc2(Fuc) at the same site. The

246

peptide backbone ions at m/z 1361.71+1 (Y1 at m/z 1564.80+1) revealed the site Asn349 with

247

sequence N349NSVDPRISNVF349-360. Sialylated complex glycans with LacdiNAc residues were

248

also present on the same site (Figure 4B). Additionally, tryptic glycopeptides with peptide

249

backbone NNSVDPR (m/z 801.40+1) was also observed and might have arisen from minor

250

contaminant of trypsin in the protease used (Table S1). In total Asn349 found to possess 11

251

different glycan structures majorly sialic/neutral fucosylated complex types followed by high

252

mannose and hybrid types with few glycan isomers (Table S1). MS/MS of goat LPO derived

253

precursor ions at m/z 960.06+3 shown to contain sialylated and fucosylated hybrid glycans with

254

sequence NeuGcGalNAc1GlcNAc1Man5GlcNAc2(Fuc) (Figure 4C). Interestingly the peptide

255

backbone mass at m/z 801.40 (Y1 at m/z 1004.48+1) matched to sequence N349NSVDPR349-355, a

256

tryptic peptide as observed in buffalo LPO. Complex type glycans was also present at Asn349

257

(Figure 4D)(m/z 987.41+3) and overall Asn349 harboured 11 different structures with mainly

258

sialic/neutral differentially fucosylated hybrid glycans (> 75%) followed by high mannose and

259

complex types (Table 1). Asn349 in bovine LPO also harbours mixture of high mannose,

260

complex/hybrid glycans (with or without fucose)13. In total we determined N-glycan pattern of

261

four glycosylation sites out of five in LPO. Table S1 summarises the overall N-glycopeptides

262

observed from chymotrypsin digestion of both buffalo and goat LPO. The sequence analysis of

263

LPO indicated that Asn258 has two proximal cysteines that form a disulfide linkage (Figure S1)

264

and to reduce these bond harsh conditions is required. In bovine LPO, Asn258 site found to

265

harbour glycans similar to Asn34913. The summary of types of N-glycans identified on individual

266

site in buffalo and goat LPO is presented in Table 2.


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267

In case of bovine LPO the glycan composition was largely determined by MS analysis13.

268

Although the glycan composition of buffalo and goat LPO was comparable to bovine LPO, the

269

lack of information on the arrangements of monosaccharides pose an ambiguity over type of

270

glycans and isomers reported for bovine. To the best of our knowledge the glycan pattern of

271

human LPO has not been explored. The characteristic feature of complex and hybrid N-glycans

272

in buffalo and goat LPO is the presence of terminal LacdiNAc residues. LacNAc moiety that is

273

ubiquitous was also observed but only in buffalo LPO glycans. N-glycans with LacdiNAc

274

termini has limited occurrence in vertebrates and are found in glycans of human and bovine

275

MFGM28, bovine whey18, rat prolactin and kidney epithelial cells29. Complex and hybrid N-

276

glycans termini are usually extended by the addition of NeuAc/NeuGc, Fuc, Gal and GlcNAc

277

residues3. Buffalo LPO complex and hybrid glycans comprised mainly Neu5Ac moiety attached

278

either to LacNAc mostly through α2-3/α2-6 linkages30 or LacdiNAc through α2-6 linkage29, 30.

279

Similar structural motif is also observed in glycans of human and bovine MFGM28. But glycans

280

of bovine whey18, buffalo MFGM31, goat and donkey lactoferrin26, 32 are largely composed of

281

mixture of Neu5Ac/Neu5Gc attached mainly to LacNAc and to some extent LacdiNAc. Goat

282

LPO complex and hybrid glycans comprised only LacdiNAc residues that are exclusively linked

283

by Neu5Gc indicates the significant difference from buffalo LPO. Another important feature of

284

glycans is the presence of isomers which contributes to structural complexity in milk

285

glycoproteins18. In buffalo LPO, isomeric complex glycans were observed due to difference in

286

Neu5Ac connectivity either to terminal Gal or GalNAc residues (Table S1). Glycans differing in

287

monosaccharide composition can also contribute to isomer species as observed in goat LPO

288

(Table S1).



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289

Although distinct complex and hybrid N-glycans were found in buffalo and goat LPO on

290

consensus tripeptide motifs some common features on glycosylation mechanism was identified

291

among bovine, buffalo and goat. A similar N-terminal glycopeptide confirmed the cleavage of

292

propeptide (100 residues including signal peptide) giving rise to mature LPO (612 residues). The

293

differential occupancy of Asn112 with only non-fucosylated glycans, presence of majorly high

294

mannose on Asn222 as well as fucosylated glycans on Asn6 and Asn349 was observed. Thus the

295

present study for the first time provided novel insights on LPO from different sources on N-

296

terminal processing, micro- and macro heterogeneity, glycan composition, sequence and isomers.

297

This would contribute in further understanding the role of glycans in LPO functionalities as well

298

as their bioactivities to make LPO an efficient bioactive in various applications.

299

Conflict of Interest: None declared

300

ACKNOWLEDGEMENTS

301

We thank Director, CSIR-CFTRI, Mysuru for the support and encouragement. We are grateful

302

to Priyanka and Dr. Navin Kumar Rastogi, Food Engineering Department, CSIR-CFTRI for

303

helping in enzyme assay. We thank Central Instrument Facility and Service, CSIR-CFTRI for

304

mass spectrometry experiments. The work was carried out with funding from CSIR-CFTRI

305

(MLP-0207).

306

ABBREVIATIONS

307

LPO,

308

chromatography; ESI, electro spray ionization; Q-TOF MS, quadrupole time of flight mass

309

spectrometer ; MS/MS, tandem mass spectrometry; Neu5Gc, N-Glycolylneuraminic acid;

lactoperoxidase;

RP,

reverse

phase;

UHPLC,


ultra-high

performance

liquid

Page 15 of 29


310

Neu5Ac, N-Acetylneuraminic acid; Man, mannose; Glc, glucose; Gal, galactose; GlcNAc, N-

311

acetyl glucosamine; GalNAc, N-acetyl galactosamine; Fuc, fucose; N-glycans, N-linked glycans.

312

ASSOCIATED CONTENT

313

Table S1: Total N-glycopeptides identified from buffalo and goat LPO in this study.

314

Figure S1. Multiple sequence alignment of human, goat, bovine, buffalo mature LPO. The N-

315

glycosylation sites are highlighted in yellow.

316

Figure S2. SDS PAGE analysis of goat LPO. Lane 1: purified buffalo LPO, Lane 2: protein

317

molecular weight marker, Lane 3: purified goat LPO.

318

Figure S3. Mascot search results for purified buffalo (A) and goat (B) LPO. The peptide without

319

glycans at Asn112 is underlined.

320

REFERENCES

321

1. van Hooijdonk, A.C.; Kussendrager, K.D.; Steijns, J.M. In vivo antimicrobial and antiviral activity of

322

components in bovine milk and colostrum involved in non-specific defence. Br. J. Nutr. 2000, 84, S127-

323

134.

324

2. O'Riordan, N.; Kane, M.; Joshi, L.; Hickey, R.M. Structural and functional characteristics of bovine

325

milk protein glycosylation. Glycobiology. 2014, 24, 220-236.

326

3. Stanley, P.; Taniguchi, N.; Aebi, M. N-Glycans. In Essentials of Glycobiology, 3rd ed., Varki, A.;

327

Cummings, R.D.; Esko, J.D., et al., Ed.; Cold Spring Harbor Laboratory Press: New York, 2017; Chapter

328

9.

329

4. Zacchi, L.F.; Schulz, B.L. N-glycoprotein macroheterogeneity: biological implications and

330

proteomic characterization. Glycoconj. J. 2016, 33, 359-76.



331

5. Reiter, B.; Harnulv, G. Lactoperoxidase antibacterial system: Natural occurrence, biological functions

332

and practical applications. J. Food Prot. 1984, 47, 724–732.

333

6. Boots, J.W.; Floris, R. Lactoperoxidase from catalytic mechanism to practical applications. Int. Dairy

334

J. 2006, 16, 1272–1276.

335

7. Haddain, M.S.; Ibrahim S.A.; Robinson, R.K. Preservation of raw milk by activation of the natural

336

lactoperoxidase systems. Food Control. 1996, 7, 149–152.

337

8. Sisecioglu, M.; Kirecci, E.; Cankaya, M.; Ozdemir, H.; Gulcin, I.; Atasever, A. The prohibitive effect

338

of lactoperoxidase system (LPS) on some pathogen fungi and bacteria. Afr. J. Pharm. Pharmacol. 2010,

339

4, 671–677.

340

9. Jyoti, S.; Shashikiran, N.D; Reddy, V.V. Effect of lactoperoxidase system containing toothpaste on

341

cariogenic bacteria in children with early childhood caries. J. Clin. Pediatr. Dent. 2009, 33, 299-303.

342 343

10. Seifu, E.; Buys, E.M.; Donkin, E.F. Significance of the lactoperoxidase system in the dairy

344

industry and its potential applications: a review. Trends Food Sci. Technol. 2005, 16, 137–154.

345 346

11. Cals, M.M.; Mailliart, P.; Brignon, G.; Anglade, P.; Dumas, B.R. Primary structure of bovine

347

lactoperoxidase, a fourth member of a mammalian heme peroxidase family. Eur. J. Biochem. 1991, 198,

348

733-739.

349 350

12. Shin, K.; Hirotoshi H.; Bo, L. Purification and quantification of lactoperoxidase in human milk with

351

use of immunoadsorbents with antibodies against recombinant human lactoperoxidase. Am. J. Clin. Nutr.

352

2001, 73, 984–989.

353

13. Wolf, S.M.; Ferrari, R.P.; Traversa, S.; Biemann, K. Determination of the carbohydrate composition

354

and the disulfide bond linkages of bovine lactoperoxidase by mass spectrometry. J. Mass Spectrom. 2000, 16 ACS Paragon Plus Environment

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35, 210-217.

356

14. Singh, A.K.; Singh, N.; Sharma, S.; Singh, S.B.; Kaur, P.; Bhushan, A.; Srinivasan, A.; Singh, T.P.

357

Crystal structure of lactoperoxidase at 2.4 Å resolution. J. Mol. Biol. 2008, 376, 1060-1075.

358

15. Sheikh, I.A.; Singh, A.K.; Singh, N.; Sinha, M.; Singh, S.B.; Bhushan, A.; Kaur, P.; Srinivasan, A.;

359

Sharma, S.; Singh, T.P. Structural evidence of substrate specificity in mammalian peroxidases: structure

360

of the thiocyanate complex with lactoperoxidase and its interactions at 2.4 Å resolution. J. Biol. Chem.

361

2009, 284, 14849-14856.

362

16. Bode, L. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology. 2012, 22,

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1147–1162.

364

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

365

Endo-beta-N-acetylglucosaminidases from infant gut-associated bifidobacteria release complex N-glycans

366

from human milk glycoproteins. Mol. Cell Proteomics. 2012, 11, 775–785.

367

18. Nwosu, C.C.; Aldredge, D.L.; Lee, H.; Lerno, L.A.; Zivkovic, A.M.; German, J. B.; Lebrilla, C.B.

368

Comparison of the human and bovine milk N-glycome via high-performance microfluidic chip liquid

369

chromatography and tandem mass spectrometry. J. Proteome. Res. 2012, 11, 2912-2924.

370

19. Nandini, K.E.; Rastogi, N.K. Integrated downstream processing of lactoperoxidase from milk whey

371

involving aqueous two-phase extraction and ultrasound-assisted ultrafiltration. Appl. Biochem.

372

Biotechnol. 2011, 163, 173-185.

373

20. Borzouee, F.; Mofid, M.R.; Varshosaz, J.; Samsam Shariat, S.Z.A. Purification of lactoperoxidase

374

from bovine whey and investigation of kinetic parameters. Adv. Biomed. Res. 2016, 5, 189.

375

21. Gnanesh Kumar, B.S.; Surolia, A. Site specific N-glycan profiling of NeuAc(α2-6)-Gal/GalNAc-

376

binding bark Sambucus nigra agglutinin using LC-MSn revealed differential glycosylation. Glycoconj.

377

J. 2016, 33, 907-915. 17 ACS Paragon Plus Environment


378

22. Stavenhagen, K.; Plomp, R.; Wuhrer, M. Site-specific protein N- and O-glycosylation analysis by a

379

C18-Porous graphitized carbon-liquid chromatography-electrospray ionization mass spectrometry

380

approach using pronase treated glycopeptides. Anal. Chem. 2015, 87, 11691-11699.

381

23. Wuhrer, M.; Catalina, M.I.; Deelder, A.M.; Hokke, C.H. Glycoproteomics based on tandem mass

382

spectrometry of glycopeptides. J. Chromatogr. B. 2007, 849, 115-28.

383

24. Nwosu, C.C.; Seipert, R.R.; Strum, J.S.; Hua, S.S.; An, H.J.; Zivkovic, A.M.; German, B.J.; Lebrilla,

384

C.B. Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures. J

385

Proteome. Res. 2011, 10, 2612-2624.

386

25. Watanabe, S.; Varsalona, F.; Yoo, Y.C.; Guillaume, J.P.; Bollen, A.; Shimazaki, K.; Moguilevsky, N.

387

Recombinant bovine lactoperoxidase as a tool to study the heme environment in mammalian peroxidases.

388

FEBS Lett. 1998, 441, 476-479.

389

26. Le Parc, A.; Dallas, D.C.; Duaut, S.; Leonil, J.; Martin, P.; Barile, D. Characterization of goat milk

390

lactoferrin N-glycans and comparison with the N-glycomes of human and bovine milk. Electrophoresis.

391

2014, 35, 1560-1570.

392

27. Nwosu, C.C.; Strum, J.S.; An, H.J.; Lebrilla, C.B. Enhanced detection and identification of

393

glycopeptides in negative ion mode mass spectrometry. Anal. Chem. 2010, 82, 9654-9662

394

28. Wilson, N.L.; Robinson, L.J.; Donnet, A.; Bovetto, L.; Packer, N.H.; Karlsson, N.G. Glycoproteomics

395

of milk: differences in sugar epitopes on human and bovine milk fat globule membranes. J Proteome.

396

Res. 2008, 7, 3687-3696.

397

29. Stanley, P.; Cummings, R.D. Structures common to different glycans. In Essentials of Glycobiology

398

3rd ed., Varki, A.; Cummings, R.D.; Esko, J.D., et al., Ed.; Cold Spring Harbor Laboratory Press: New

399

York, 2017; Chapter 14.


Page 18 of 29

Page 19 of 29


400

30. Varki, A.; Schnaar, R.L.; Schauer, R. Sialic acids and other nonulosonic acids. In Essentials of

401

Glycobiology 3rd ed., Varki, A.; Cummings, R.D.; Esko, J.D., et al., Ed.; Cold Spring Harbor Laboratory

402

Press: New York, 2017; Chapter 15.

403

31. Brijesha, N.; Nishimura, S.I.; Aparna, H.S. Comparative Glycomics of Fat Globule Membrane

404

Glycoconjugates from Buffalo (Bubalus bubalis) Milk and Colostrum. J Agric. Food Chem. 2017, 65,

405

1496-1506.

406

32. Gallina, S.; Saletti, R.; Cunsolo, V.; Muccilli, V.; Foti, S.; Roepstorff, P.; Rasmussen, M.I. Site-

407

specific glycosylation of donkey milk lactoferrin investigated by high-resolution mass spectrometry.

408

Amino Acids. 2016, 48, 2799-2808.

409

Figure legends:

410

Figure 1. N-glycans at Asn6. MS/MS of N-glycopeptides precursor ions from buffalo LPO at (A) m/z

411

1230.58+3 and (B) m/z 1200.89+3 indicating sialylated fucosylated hybrid glycans with Neu5Ac linking to

412

LacdiNAc and neutral fucosylated complex glycans comprising terminal LacNAc and LacdiNAc

413

moieties. MS/MS of goat LPO derived N-glycopeptides precursor ions at (C) m/z 1187.55+3 and (D) m/z

414

1235.90+3 corresponding to neutral fucosylated hybrid glycans with LacdiNAc moiety and sialylated

415

fucosylated hybrid glycans with Neu5Gc attached to LacdiNAc residue respectively. Ions at m/z 495.19

416

[Neu5Ac+HexNAc+H]+1 and m/z 698.27 [Neu5Ac+2HexNAc+ H]+1 in (D) indicate the presence of

417

glycan isomer (NeuAc1GalNAc1GlcNAc1Man5GlcNAc2). The common ions in all MS/MS at m/z

418

1993.04+1 matches to peptide sequence DTTLTN6VTDPSLDLTAL1-17 + GlcNAc in both the species.

419


420

1195.55+3 and (B) m/z 1167.86+3 indicating sialylated non-fucosylated complex glycans with Neu5Ac

421

linking to LacdiNAc and LacNAc moieties respectively. The ions at m/z 1789.93+1 in (A) and (B)

422

matches to peptide sequence LDEDGVLDQN112RSLL103-116 + GlcNAc. MS/MS of goat LPO derived N-

423

glycopeptides precursor ions at (C) m/z 1188.07+4 and (D) m/z 1208.84+4 corresponding to sialylated 19 ACS Paragon Plus Environment


424

(Neu5Gc) hybrid and complex glycans. The ions at m/z 1511.32+2/1007.55+3 in (C) and (D) matches to

425

peptide sequence AREVSNKIVGYLDDEGVLDQN112RSLL92-116 + GlcNAc.

426

Figure 3. N-glycans at Asn222. MS/MS of N-glycopeptide precursor ions at m/z 1164.02+2 derived from in

427

buffalo and goat LPO indicating high mannose glycans. The ions at m/z 989.55+1 matching to peptide

428

sequence RN222LSSPL221-227 + GlcNAc.

429


430

1141.84+3 and (B) m/z 1168.85+3 indicating sialylated fucosylated complex glycans with Neu5Ac linking

431

to LacNAc and LacdiNAc residues, respectively. The ions at m/z 1564.80+1 matched to peptide

432

N349NSVDPRISNVF349-360 + GlcNAc. Additional ions at m/z 407.17, 495.19 and 698.27 might have arise

433

from hybrid glycan (NeuAc1GalNAc1GlcNAc1Man5GlcNAc2(Fuc) at the same site. MS/MS of goat LPO

434

derived N-glycopeptides precursor ions at (C) m/z 960.06+3 and (D) m/z 987.41+3 corresponding to

435

sialylated fucosylated hybrid and complex glycans. Ions at m/z 1004.48+1 matches to peptide sequence

436

N349NSVDPR349-355+ GlcNAc (see text).


Page 20 of 29

Page 21 of 29


A

B 495.1969

2400

366.1491 5500

698.2823

2300 2200

5000

2100 2000

4500

407.1764

1900 1800

4000

1700 1600

DTTLTNVTDPSLDLTAL

Intensity

1400

Intensity

1500 204.0923

1300 407.1782

1200 1100 900

Pep

M+3H3+

800

2000

PSLDLTAL 829.4918

699.2863 528.2081 690.2662

366.1504

700 600

1181.5528

860.3397

Pep 997.5295+2 1176.2241

300 200

1000

Pep Pep 1597.7687+2 +1 1597.2644 1790.9617

100 300

400

500

2900

600

700

800

900

1000 1100 Mass/Charge

1200

1300

1400

1500

1600

1700

1800

200

1900

300

400

500

600

700

800

900

Pep Pep 1993.0460 Pep 1597.7607+2 1790.9530+1 1699.8057+2

Pep 1055.4042

1443.6913+2


1200

1300

1400

1500

1600

1700

1800

1900

D

407.1767 204.0920

2600

3800

2500

714.2741

3600

2400 2300

3400

2200

3200

2100

511.1899

3000

2000 1900

2800

1800

2600

DTTLTNVTDPSLDLTAL

1700 1500

M+3H3+

1187.5578

1400 1300

2200

1800

1100

1600 Pep 1995.0491+1

1000 900 408.1800

700

829.4880

600 690.2628

852.3185+1

569.2308

+3 1084.84571133.5372 +3 1014.3747+1

200

Pep

Pep Pep 1577.7475+2 Pep 1496.7156+2 1423.6865+2

1400

1342.6552+2

0

0

400

500

600

700

800

900


1200

1300

1400

1500

1600

528.2045

829.4875 690.2622

1700

852.3209 495.1939

1186.8767+3 Pep +2 944.5167 997.5239 1055.4053

876.3312

698.2809

400 200 1800

1900

1993.0426+1

PSLDLTAL 715.2765

366.1490

600

100 300

290.0942

800 1993.0397

Pep 1679.2869+2

Pep

M+3H3+ 1235.9032+3 1235.5707

1000

1188.2252

528.2055 205.0949

300

200

407.1762

1200

PSLDLTAL

366.1491

500

204.0919

2000

1200

800

DTTLTNVTDPSLDLTAL

2400

1600

Intensity

Intensity

829.4881+1

731.2906+2

2800

400

1994.0468+1

PSLDLTAL 528.2054 569.2335

500 0

0

Pep

1200.8983 1201.5679

1995.0561

Pep Pep 1415.1893+2 1496.2219+2 Pep 1342.1618+2

852.3236

400

2700

M+3H3+

1500

500

C

2500

1230.2438

274.1004

DTTLTNVTDPSLDLTAL

3000

1993.0497+1

1230.5813

1000

204.0920

3500

200

Figure 1


300

400

500

600

700

800

900

1000 1100 1200 Mass/Charge

Pep 1415.1851+2 Pep Pep 1342.1515+2 1496.2166+2

Pep 1597.7511+2

1300

1400

1500

1600

1700

Pep 1790.9482+1 1800

1900


B

A 407.1766

2300

4000

2100

3800

2000

3600

1900 1800

495.1952

204.0920

3400

698.2796

M+3H3+

1700

3200

1195.5519

3000

1600

LDEDGVLDQNRSLL

2800

1195.2152 Pep

1300

1443.6884+2

1200 1100 1000

2400

657.2503

2200

1167.8640

1600

800 700 600

Pep

699.2836 292.1107 408.1793

500 400

Pep

496.1982

300

366.1482

1362.1504

895.4691+2

200

300

400

500

600

700

800

1000 800

900

C


1200

1300

1400

1500

1600

1700

1800

0

1900

1188.3291

714.2744

1345.6762

AREVSNKIVGYLDEEGVLDQNRSLL

204.0918

Intensity

700 290.0946 407.1762 500

Pep Pep 1413.7061+3

400 1291.9919+3 512.1910

308.1049

Pep 1481.4022+3 1481.7312

1187.5754 408.1791

100

529.2019 513.1956 409.1787 200

300

400

500

600

852.3207

Pep 1007.8835+3 1007.5506 1111.3020+4

1511.3201+2

800

900

1000 1100 1200 Mass/Charge

Pep

1612.8743+2

876.3337 700

400

500

600

700

800

900


1300

1200

1300

1488.1954 1400

1500

1600

1700

1800

1900

M+4H4+ 1208.8452+4

1400

1500

1600

2000 1900 1800 1700 1600 1500 1400

AREVSNKIVGYLDEEGVLDQNRSLL

1700

Pep Pep 1774.9119+21856.4556+2 1937.4836+2 1800

1900

407.1773

204.0924

Pep 511.1914

1300 1200 1100 1000

1374.0356

Pep Pep

714.2758 1441.3955+3 1441.7317

290.0949

Pep

715.2804

500 400 300 200 100 0

1209.0960+4 Pep 1373.7006+3

900 800 700 600

1413.3706

715.2797

200

300

1240.5968+2

2400 2300 2200 2100

Pep 1346.0121+3

300

200

1159.5671 997.5142+2

731.2867

472.1824

Pep 1568.7247+2 Pep 1790.9321+1 1605.7420+2

Pep 819.3093 895.9687+2 Pep

2700 2600 2500

1000

600

528.2029

D

1188.0784+4

800

601.3099

200

1100

900

367.1518

204.0917

Pep

Pep 1342.1429+2

400

M+4H4+

511.1900

274.0982

600

1240.0968+2

861.3447 794.4236

100

1423.6752

1200

1342.6399

860.3327 690.2588 700.2852 569.2347

200

Pep

1545.2313+2 Pep +1 1589.2386+2 1789.9316 1589.7355 1790.9341 1690.7839+2

Pep 1362.6569+2

1168.5373

1400

Pep

274.0988

Pep 1423.1706+2

M+3H3+

2000 1800

900

0

LDEDGVLDQNRSLL

2600

1400

Intensity

Intensity

1500

0

366.1483

4200

2200

Intensity

Page 22 of 29

408.1810

512.1940

1131.5586+4

Pep

1319.6804+3 1509.0902+3

Pep

1543.7638+3

876.3342 1008.2204+3 569.2335 200

Figure 2


300

400

500

600

1008.5554 700

800

900


1200

1300

1400

1500

1600

Pep 1958.4927+2 1856.9597+2 1775.9321+2

1700

1800

1900

Page 23 of 29


M+2H2+

1164.5029+2 8000

Pep 989.5471+1

7500 7000 6500 6000

Intensity

5500 5000 4500 4000

990.5516

RNLSSPL

3500

1083.4914+2

1165.9132

3000 2500

Pep

366.1480 2000

204.0917

1500

325.1208 528.2034

229.1591

500 200

300

1166.3917

758.8778+2 839.9051

+2

1338.4880+1 1192.6333+1

677.8486+2

PL

1000

0

920.9343+2

1354.6914+1

487.1762 367.1518 400

500

1516.7410+1 600

700

800

900


1200

1300

Figure 3


1400

1500

1678.8081+1 1840.8635+11936.8313+1

1600

1700

1800

1900


Page 24 of 29

B

A 9500

366.1489

2000

8500

1900

8000

1800

7500

1700

7000

1600

204.0927

NNSVDPRISNVF

1200

657.2516

4500

1169.1957

1100

Pep 1404.1514+2

1000 900

4000

800

3500

700

M+3H3+

3000 274.0991 204.0915

Pep 1383.1364+2

1141.1769

1500 528.2055 690.2612

407.1765

500

500 292.1100

400

1383.6403

Pep 1302.6101+2

698.2781 PRISNVF 832.4896

495.1937

1000

200

300

400

500

600

700

800

900


1200

1300

PRISNVF 832.4875+1 860.3364

200

1400

1500

1600

1700

Pep Pep +2 1505.6951+2 1550.2065 1549.6995 Pep 1710.8623+1 Pep

100

1800

1900

200

300

400

500

600

700

800

900

D

511.1878

204.0909

1100

Pep 1323.1217+2

496.1993

300

Pep Pep Pep 1564.8033+1 1710.8639+1 1361.7121+1

0

0

699.2827

274.0992

600

1141.8459

2000

1200

1168.8595

698.2791

1300

NNSVDPRISNVF

5500

Intensity

Intensity

1400

6000

5000

C

M+3H3+

495.1949

1500

6500

2500

407.1767

2100

9000

1600


1200

1300

1400

1500

1600

1700

1800

1900

407.1748

1500

714.2723

1400

1000

M+3H3+

800

960.0679

407.1754

NNSVDPR Pep

600 715.2756 500

960.7346

512.1905

300

Pep 1184.5061+2

500

1185.0046

690.2622 529.2043

100

300

Pep 1286.0440+2

Pep 801.4048

1370.6268 0

200

300

400

500

600

700

800

900


987.4192

1200

1300

1400

Pep 1531.6879+1 1500

1600

1700

1839.7878+1 1800

1900

Pep 1225.53+2 Pep 1042.9578+2 1004.4851 1150.5449+1

512.1908

272.1777 308.1035

716.2769

409.1792 200

Figure 4


300

Pep 1277.5355+2

715.2734

408.1781

100 0

987.7538

290.0932

200

Pep

Pep 1693.7309+1

Pep

Pep 1123.9910+2

400

528.2027

200

M+3H3+

714.2720

600

1150.5484+1

308.1035

511.1880

900

700

1083.4642

400

1000

800

960.4033

366.1474

NNSVDPR

1100

Pep 1082.9636+2

290.0932

700

204.0911

1200

Intensity

900

Intensity

1300

Pep 1004.4870+1

400

500

Pep 1327.0755+2

700

800

1840.8065+1 1677.7449+1 1694.7438+1

1005.4887

1531.6836+1

801.4012+1 600

Pep Pep 1379.0869+2

900


1200

1300

1400

1532.6847 1500 1600 1700

1800

1897.8303+1

1900

Page 25 of 29


Table 1: Summary of glycans sequence and their relative abundancea at individual site in buffalo and goat LPO

Buffalo LPO

Goat LPO

Sites abundance

Glycan composition

% abundance

Glycan composition

%

N6VT

HexNAc4Hex3HexNAc2(dHex)(C)

33

HexNAc2Hex5HexNAc2(H)

9


8


4

HexNAc2Hex4HexNAc2(dHex)(H)

22


20


17


15

NeuAcHexNAc4Hex3HexNAc2(dHex)(C)

11

NeuGcHexNAc2Hex5HexNAc2(H)

10

NeuAcHexNAc2Hex4HexNAc2(dHex)(H)

9

NeuGcHexNAc4Hex3HexNAc2(dHex)(C)

7

NeuGcHexNAc2Hex4HexNAc2(dHex)(H)

25


10

N112RS b

Hex5HexNAc2(HM)

6


16

HexNAc2Hex5HexNAc2(H/C)

15


10

HexNAc3Hex4HexNAc2(C)

19


48


39


3

NeuAcHexNAc2Hex5HexNAc2(C)

7


2


12

NeuGcHexNAc4Hex3HexNAc2(C)

21


2



N222LS

Page 26 of 29

Hex8HexNAc2(HM)

8

Hex9HexNAc2(HM)

7

Hex7HexNAc2(HM)

40

Hex8HexNAc2(HM)

6

Hex6HexNAc2(HM)

34

Hex7HexNAc2(HM)

30

Hex5HexNAc2(HM) 42

9

Hex4HexNAc2

Hex6HexNAc2(HM) 9

Hex5HexNAc2(HM)

7

N349NS

Hex4HexNAc2

8

Hex5HexNAc2(HM)

7

Hex5HexNAc2(HM)

4

Hex6HexNAc2(HM)

1

Hex6HexNAc2(HM)

8

HexNAc2Hex5HexNAc2(dHex)(C/H)

16


5


14


11


3


2

NeuAcHexNAc1Hex6HexNAc2(dHex)(H)

1


13

NeuAcHexNAc2Hex5HexNAc2(dHex)(C/H) 44


3


8


23


6


4


17

NeuGcHexNAc4Hex3HexNAc2(dHex)(C)

10

_________________________________________________________________________________________________________ _________ a relative

abundance was calculated based on peak area from EIC of each glycoform 26 ACS Paragon Plus Environment

Page 27 of 29


b

NRS showed 65 % and 40 % occupancy in buffalo and goat LPO, respectively C- complex, H- hybrid, HM- high mannose



Page 28 of 29

Table 2: Comparison of site-specific glycan types observed in buffalo and goat LPO _________________________________________________________________________________________________ _______ Type

Buffalo LPO

Goat LPO

_________________________________________________________________________________________________ _______ N6

N 112

N 222

N 349

High mannose

-

+

+

+

Neutral complex/hybrid

-

+

-

Neutral fucosylated complex/hybrid

+

-

Sialylated complex/hybrid

-

Sialylated fucosylated complex/hybrid

+

N 112

N 222

N 349

-

-

+

+

-

+

+

-

+

-

+

+

-

-

+

+

-

-

+

+

-

+

-

-

+

+

-

-

+


N6

Page 29 of 29


Buffalo and goat whey

Ammonium sulfate fractionation

Buffalo LPO

Cation exchange chromatography

Goat LPO

Proteolysis

RP-UHPLC-HRMS

MS Interpretation

N-glycopeptides

Graphical abstract


Comparative Site-Specific N-Glycosylation Analysis of

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