Capillary Electrophoresis Analysis of Bovine Milk Oligosaccharides

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Food and Beverage Chemistry/Biochemistry

Capillary electrophoresis analysis of bovine milk oligosaccharides permits an assessment of the influence of diet and the discovery of nine abundant sulfated analogues Sara Vicaretti, Nina Mohtarudin, Alexander Garner, and Wesley F. Zandberg J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01041 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 13, 2018

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Journal of Agricultural and Food Chemistry

Capillary Electrophoresis Analysis of Bovine Milk Oligosaccharides Permits an Assessment of the Influence of Diet and the Discovery of Nine Abundant Sulfated Analogues. Sara D Vicaretti1, Nina A Mohtarudin2, Alexander M Garner2, and Wesley F Zandberg1* 1

Department of Chemistry and 2Biochemistry, The University of British Columbia, Okanagan

* Corresponding author contact details: Wesley F Zandberg Tel: 250-807-9821; Fax: 250-807-8001; Email: [email protected]

1 ACS Paragon Plus Environment


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1

Abstract.

2

Bovine milk oligosaccharides (BMOs), like their analogues in human milk, have important

3

prebiotic functions. Environmental factors have previously been linked to variation in BMO

4

structures, thus to test the hypothesis that the bovine diet may lead to these changes in relative

5

BMO abundances, a rapid capillary electrophoresis (CE)-based work flow was developed to

6

profile the BMOs extracted from the milk of cows fed distinctly different diets. Over the first

7

week of lactation, few significant differences were observed between the different diet groups,

8

with the dominant changes being clearly linked to lactation period. CE analyses indicated the

9

presence of ten unusually anionic BMOs which were predicted to be phosphorylated and sulfated

10

species. Nine unique sulfated BMOs were detected by high-resolution accurate mass

11

spectrometry, none of which have been previously described in bovine milk. The biosynthesis of

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these was in direct competition with 3’-sialyllactose, the most abundant BMO in bovine milk.

13 14

Keywords: milk; sulfated oligosaccharides; capillary electrophoresis; mass spectrometry; diet.


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Introduction

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Oligosaccharides are among the most abundant components of human milk, being found

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in both their free forms, typically with the disaccharide lactose found at the reducing end, and

18

conjugated to other biomolecules such as milk proteins and lipids. The abundance of free,

19

unconjugated human milk oligosaccharides (HMOs) is especially notable in light of the fact that

20

these are indigestible to neonates. Though not directly used to meet the newborns’ caloric needs,

21

HMOs are important bioactive molecules with numerous health-promoting roles.1,2 For example,

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HMOs function as prebiotics that encourage the colonization of the infant gastrointestinal (GI)

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tract with a protective microbiome.3,4 In addition, HMOs often bear glycoepitopes that are also

24

observed on the protein-linked oligosaccharides comprising the mucus lining the gut, permitting

25

them to serve as soluble decoys capable of blocking the attachment of pathogens to the intestinal

26

epithelium. Recently, it has been demonstrated that HMOs may bind to lectins expressed on the

27

surface of dendritic cells5–7 in the intestinal epithelium, and in this capacity they may directly

28

influence inflammatory processes8 critical to both the short- and long-term health of neonates.

29

The ability of HMOs to engage cell surface lectins, coupled with evidence of systemic

30

absorption from the GI tract,9 is also consistent with observations that these compounds have

31

important functions beyond their roles in the GI tract.10

32

The numerous biological functions of HMOs are clearly dependent on their specific

33

chemical structures. To date, over 250 HMOs have been detected (although many still await

34

structural identification),1,2 most of which are based on a lactose core structure (Gal-β1,4-Glc,

35

where Gal and Glc are the monosaccharides D-galactose and D-glucose, respectively) that has

36

been further elongated with Gal, N-acetyl-D-glucosamine (GlcNAc), L-fucose and 5-N-acetyl-D-

37

neuraminic acid (Neu5Ac) residues (Figure S1). Like HMOs, bovine milk oligosaccharides



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(BMOs) are composed of the same five monosaccharides, in addition to the non-human,

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mammal-specific monosaccharide 5-N-glycolyl-D-neuraminic acid (Neu5Gc; Figure S2).

40

Relative to HMOs, BMOs are found at roughly 20-fold lower absolute concentrations, and have

41

higher relative levels of acidic structures containing Neu5Ac/Neu5Gc in preference over the

42

fucosylated analogues that are more prevalent in human milk. To date, over 50 distinct BMOs

43

have been detected,11,12 a number of which are structurally identical to HMOs. Although BMOs

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have not received as much research attention, a clearer understanding of their structures and the

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factors affecting their biosynthesis is warranted given that they may confer HMO-like health

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benefits to milk and dairy product consumers13 and have been established as viable additives to

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infant formulas, that are otherwise nearly devoid of HMOs/BMOs, prompting efforts to recover

48

BMOs from dairy processing streams.14

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The dependence on the health-promoting effects of milk oligosaccharides on their

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structures, and the notable variation in structures observed between women, not to mention the

51

differences between human and bovine milk, has prompted research into the biochemical basis

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for milk oligosaccharide variation and the subsequent impact on infant health.15,16 It is known

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that genetic factors are responsible for much of the observed variation in the relative HMO

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concentrations in the milks collected from healthy mothers; these are thought to have a

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significant impact on infant.4,13,16,17 Genetic sources of variation in milk oligosaccharides from

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other mammals is less clear. Sundekilde et al. have identified differences in the BMO pools in

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milk from Jersey and Holstein-Friesian cows, with the former producing higher relative amounts

58

of Fuc- and Neu5Ac-containing structures.18 Meanwhile, the non-genetic factors influencing

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milk oligosaccharide composition in either humans or cows still await a more rigorous

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characterization. It is known that the levels of both HMOs15 and BMOs19,20 decrease during the


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course of lactation. Liu et al. have recently demonstrated that BMO levels exhibit a seasonal

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variability, with generally higher levels observed in the autumn.21 A similar trend has also been

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observed in human milk, with total HMO levels significantly decreasing in the milk of mothers

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when food resources are more scarce.16 These studies, paired with evidence of clear variations

65

among HMO levels observed between ethnically (and presumably genetically) similar women

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living in differing locations,22 suggests that the environment may play an important role in

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regulating the biosynthesis of milk oligosaccharides. We hypothesize that the maternal diet may

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represent a significant non-genetic factor impacting both HMO and BMO biosynthesis, thus

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explaining the seasonal and geographical trends noted to date. Such a dietary link would also be

70

consistent with reports indicating that over nourishment/obesity,1 malnourishment13 and

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hyperglycemia1 all impact the structures and/or relative concentrations of HMOs.

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To test the hypothesis that the maternal diet impacts milk oligosaccharide biosynthesis

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we compared the BMOs produced by dairy cows fed different diets over the first week of

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lactation. It was reasoned that any correlations would be more readily detectible in dairy cows

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since these lack the obvious genetic differences observed amongst HMOs, and since, unlike

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people, dairy cattle within a single farm are all fed essentially identical diets. A recent study

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comparing pasture-only-fed cows on organic or conventional diets has indicated that the bovine

78

diet may have had a slight impact on the levels two (of 11 analyzed) BMOs 23; we reasoned that

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BMO differences would be more readily apparent by comparing cows fed grass alone to those

80

whose diets also included corn and grain. A second aim of this research was to develop a rapid

81

analytical work flow permitting the accurate investigation of the biosynthetic relationships

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between BMOs (or HMOs). In this respect, it should be noted that while mass spectrometry

83

(MS) is one of the most sensitive and chemically-informative techniques available for



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BMO/HMO analysis, the vastly different response factors for different oligosaccharides—for

85

example, neutral versus acidic (i.e., Neu5Ac/Neu5Gc-containing) species—complicates a clear

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assessment of the biosynthetic relationships between BMOs present within a single sample,

87

especially in the absence of suitable stable isotopic analogues.24 A solution to this challenge is to

88

analyze these compounds after labelling their reducing ends with fluorophores (or

89

chromophores) to permit their detection by optical techniques that are insensitive to the BMO

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structures4,11,22. Thus, a capillary electrophoresis (CE) method employing laser-induced

91

fluorescence (LIF) detection was used here to rapidly (within 8 min) establish the relative

92

quantities of 34 BMOs shared in common with all the individual milk samples collected; these

93

BMOs were also analyzed in pooled samples collected from three dairy farms and a range of

94

processed liquid milk samples sold by local grocery stores. High-performance liquid

95

chromatography (HPLC) coupled with high-resolution accurate mass-MS was also utilized to

96

characterize unique BMOs identified by CE-LIF, at least ten of which have not yet been

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previously described in either bovine or human milk.

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Materials and Methods

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Chemical and General Details. The following chemicals were purchased from Sigma Aldrich

100

(Saint Louis, MO, USA) and used as received: HPLC- or analytical grade acetonitrile (ACN),

101

methanol (MeOH), chloroform (CHCl3), trifluoroacetic acid (TFA), sodium acetate (NaOAc),

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sodium cyanoborohydride (NaBH3CN), dimethylsulfoxide (DMSO), acetic acid (AcOH), and

103

4,5-dimethyl-1,2-benzyldiamine (DMBA). The following chemicals were purchased from Alfa

104

Aeser or J.T. Baker (Thermo Fisher Scientfic; Tewksbury, USA; Ontario, Canada) and used as

105

received: Sodium hydroxide (NaOH), and ethanol (EtOH). All BMO and/or HMO standards

106

(Figure S1) were purchased from Dextra Laboratories Inc. (Reading, UK) or Carbosynth Ltd.


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(Compton, UK). Unless otherwise noted, 18 MΩ water was provided by a Barnstead E-Pure

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water purification system (Thermo Fisher Scientific; Waltham, MA, USA) for preparing all

109

aqueous solutions, including uHPLC mobile phases.

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(APTS) was synthesized exactly according to a published procedure25 with the exception that the

111

total reaction time was reduced from 30 h to 3 h. APTS was further purified over a graphite

112

column26 and quantified by fluorescence, using commercially-available APTS (SCIEX;

113

Washington, DC, USA) as a calibration standard. APTS was stored as a 100 mM stock in 0.9 M

114

citric acid at -20 °C. A DMBA stock solution (240 mM in 2M AcOH) was prepared freshly

115

before use. The following enzymes were purchased from New England Biolabs (Ipswich, MA,

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USA) and used exactly as described in the manufacturer’s instructions: calf intestinal alkaline

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phosphatase,

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perfringens) was obtained from Sigma-Aldrich.

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Milk Sample Collection and BMO Preparation. Bovine milk samples were collected at Grass

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Roots Dairies (Salmon Arm, B.C., Canada) and Hutley Acres Dairy (Armstrong, B.C., Canada)

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in 50 mL plastic milk testing tubes; samples were frozen immediately after collection,

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transported to the lab on ice and stored at -20 °C prior to thawing at 4 °C before extraction. Cows

123

at Grass Roots were fed exclusively grass diets; those at Hutley Acres were fed a diet consisting

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of corn and alfalfa silage, earlage, and grain. Milk from three animals per farm was collected:

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two Holsteins and a Holstein-Jersey cross (Hutley) and two milking Shorthorn and a Shorthorn-

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Holstein cross (Grass Roots). Three time points were sampled: as soon as possible post-calving

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(day 0), after 1 day (i.e., 24 h post-calving) and one week. At each farm, milk was collected from

128

sequentially freshening cows to ensure that the bovine diets were as similar as possible. A single

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sample (labelled “tank”) was also collected from the storage tank at each farm; these pooled

β-1,3-galactosidase,

and

8-aminopyrene-1,3,6-trisulfonic acid

β-hexosaminidasef.

Neuraminidase

(Clostridium



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samples were compared with another unprocessed milk sample (Riverbreeze Dairy, Armstrong,

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B.C., Canada) in addition to processed milk samples obtained from a local (Kelowna, B.C.,

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Canada) grocery store. All samples were collected in the late spring/early summer.

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BMO-rich fractions were prepared from whole milk by liquid-liquid extractions as

134

described elsewhere.12,18,19,27 A 2 mL aliquot of milk was mixed with 400 µL of 18 MΩ water

135

and centrifuged for 30 min at 15,100 × g at room temperature. The fat (top layer) was removed,

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leaving a protein- and oligosaccharide-rich bottom layer that was mixed with 2:1

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chloroform:methanol (v/v) at a 4:1 solvent:sample ratio. This mixture was centrifuged for 30 min

138

at 3,270 × g at 4 °C and the top, oligosaccharide-rich layer was removed and mixed with ethanol

139

at a 2:1 (ethanol:sample) ratio. This mixture was stored overnight at -42 °C to facilitate the

140

precipitation of any remaining proteins, which were pelleted by centrifugation (30 min, 3,270 ×

141

g, 4 °C). The supernatant was dried in vacuo using a SpeedVac Concentrator (Thermo-Fisher

142

Scientific) at ambient temperature. Further BMO purification, including the elimination of the

143

majority of the lactose present (Table S1), was performed using pre-conditioned graphitized

144

carbon SPE cartridges26 (Supelco, ENVICarb, 200 mg). Acidic- (50% ACN/0.1% TFA) and

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neutral- (20% ACN) BMO-containing SPE fractions were combined and dried in vacuo.

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Desalted BMOs were re-dissolved in 18 MΩ H2O and lyophilized in five equivalent fractions for

147

(1) determining total reducing sugar levels, (2) neutral monosaccharide analysis, (3)

148

quantification of total Neu5Ac/Neu5Gc levels, (4) BMO profiling by CE-LIF and (5) targeted

149

HPLC-MS analysis.

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BMO Analyses. (1) Reducing sugars were quantified by absorbance according to the method of

151

Mopper and Grindler.28 (2) BMOs equivalent to 10 nmol reducing ends were fortified with 5

152

nmol L-rhamnose (Rha) and D-N-acetylgalactosamine (GalNAc), as internal standards, 8 ACS Paragon Plus Environment

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lyophilized in 200 µL tubes, and subsequently hydrolyzed to their constituent monosaccharides

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using 100 µL 2 M TFA (100 °C, 2 h)29 after which they were snap frozen in liquid nitrogen and

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concentrated using a SpeedVac. Samples were re-N-acetylated29 and labelled with APTS,25 at 60

156

°C for 2 h in the dark (Figure S3). Monosaccharides were separated and quantified by CE-LIF

157

(ProteomeLab PA800; Beckman-Coulter) under normal polarity (+30 kV) using 240 mM

158

NaBO4, plus 0.1% (w/v) polyethylene glycol, pH 9.5 as the background electrolyte. All

159

monosaccharide peaks were identified by means of external standards and normalized to the Rha

160

peak. The ratios of the monosaccharides Gal, Fuc, and GlcNAc were all determined relative to

161

the single reducing end Glc residue of BMOs. (3) BMO-bound Neu5Ac and Neu5Gc were

162

quantified exactly as described in the accompanying manuscript.30 (4) BMOs were labelled with

163

APTS (16 h, room temperature) exactly as previously described (Figure S3).25,26 Labelling

164

reactions were quenched by adding 100 µL H2O before all samples were analyzed by CE-LIF

165

using reversed polarity (-30 kV) and a proprietary N-CHO separation buffer (SCIEX) as the

166

background electrolyte. All peaks not attributable to the APTS reagents were manually

167

integrated using 32 Karat (version 8.0) software. Some APTS-labelled samples treated with

168

glycosidases. In these assays 17 µL of sample were mixed with 2 µL 10x reaction buffer

169

(provided with each enzyme) and 1 µL enzyme, and incubated overnight at 37 °C after which

170

they were analyzed, without work up, by CE. (5) BMO samples were prepared for uHPLC-MS

171

analysis by first reducing them (1 M NaBH4 in 50 mM NH4OH; 2 h; 65 °C) to avoid the

172

resolution of α- and β-anomers;12 after neutralizing these reactions with AcOH, BMOs were

173

desalted by graphite SPE as described above. HPLC-MS parameters are described in Table S2.

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Data Acquisition and Statistical Analysis. The HPLC-MS data acquisition and processing was

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performed using the MassHunter Workstation software suite (Agilent Technologies), with the 9 ACS Paragon Plus Environment


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following version numbers: Data Acquisition Workstation (v B.08.00). HMO structures were

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characterized using the Agilent MassHunter Qualitative Analysis (v B.07.00, Service Pack 2)

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find-by-formula algorithm using a ±10 ppm mass window. Data reduction and statistical

179

calculations were performed using Microsoft Excel (Microsoft Corporation; Redmond, USA).

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CE peak areas were divided by their respective migration times and expressed as a

181

fraction of the total integrated area of all 34 peaks detectible in individual electropherograms,

182

excluding lactose. In several instances where peaks could not be detected, due to poor resolution

183

or low overall BMO abundance, the relevant region of baseline was manually integrated. Median

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BMO levels at differing stages of lactation (n = 6 cows) were deemed statistically significant if

185

the two-tailed Mann-Whitney U values were lower than Ucrit at P < 0.05 or 0.01. Differences

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between mean BMO levels in the two diet groups (n = 3 cows/group) were assessed using a two-

187

tailed Student’s t-test for heteroscedastic samples, with p < 0.05 considered significant.

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Associations between BMOs were determined by calculating the two-tailed Spearman rank

189

correlations for all biological replicates (N = 18; 6 cows x 3 time points) with ρcrit values above

190

0.472 or 0.600 considered significant at p = 0.05 and 0.01, respectively.

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Results and Discussion

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Monosaccharide Analysis. To initially test the hypothesis that the bovine diet impacts BMO

193

structures and levels, the monosaccharide content of BMOs extracted from milk obtained from

194

cows fed exclusively grass (gr) or those fed a more typical diet of corn silage, earlage and grain

195

(c/g) were compared (Figure 1). As previously noted,19 a consistent drop in the total levels of

196

reducing sugars (Figure 1A) was observed over the first week of lactation, although after one

197

week the average BMO levels still exceeded those detected in the bulk storage tank at each farm.

198

However, bovine diet did not appear to influence total levels of BMOs observable at any time 10 ACS Paragon Plus Environment

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point. BMOs were completely hydrolyzed to their constituent monosaccharides which were

200

fluorescently labeled with APTS and analyzed by CE (Figure 1B) permitting their levels to be

201

determined relative to the reducing end Glc; note, however, that traces or residual lactose would

202

lead to an underestimation of the relative levels of these monosaccharides. The relative levels of

203

the neutral monosaccharides Fuc, Gal, and GlcNAc (Figure 1C) remained constant during the

204

first seven days of lactation and did not vary with bovine diet. Likewise, the acidic

205

monosaccharides (Neu5Ac and Neu5Gc), which were quantified by HPLC-MS, did not vary

206

significantly between the two diet groups, although they both rapidly decreased during the first

207

week of lactation (Figure 1D). It is, however, unclear based on these data only, if the declining

208

Neu5Ac/Neu5Gc levels were due to the general decrease in total BMO levels (Figure 1A) or a

209

more specific change in BMO biosynthetic pathways, such as a reduction in the levels or

210

activities of the required Neu5Ac transferases or, conversely, an increase in a competitive

211

biosynthetic process. Nevertheless, these data indicate that grass- versus corn/grain-containing

212

bovine diets do not noticeably affect the relative levels of monosaccharides comprising BMOs.

213

Characterization of Bovine Milk Oligosaccharides by Capillary Electrophoresis. CE-LIF

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has proven to be an ideal separation technique for highly polar and/or charged analytes like

215

oligosaccharides, having previously been used to characterize HMOs,31 and porcine32 or equine33

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milk oligosaccharides. Prior to CE analysis, oligosaccharides are typically labelled at their

217

reducing ends with the highly fluorescent and charged compound APTS25,26,29 (Figure S3)

218

enabling the efficient separation of both neutral and acidic oligosaccharides, and ensuring

219

equivalent response factors across all oligosaccharide structures. Here, CE-LIF was used as a

220

screening tool to rapidly profile the intact BMOs (Figure 2). Peak areas for thirty four BMOs,

221

encompassing all those that could be reliably detected in most milk samples, were determined



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(peaks are numbered sequentially in Figure 2A). Nine individual BMOs were identified based on

223

their co-migration with authentic standards and chemical characteristics of others could be

224

deduced on the basis of their sensitivity to well-characterized exo-glycosidases (Table S3).

225

Relative BMO levels (Figure 2B and Figure S4) were determined by expressing each individual

226

peak area as a percentage of the total integrated area, i.e., the sum of all 34 peaks with the

227

exception of peak 14, which was composed of the co-migrating pair 6’SNL and lactose, traces of

228

which remained after the BMO extraction process. Six sialylated (i.e., Neu5Ac-containing)

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BMOs were detected in all samples (Table S3), the most abundant of which was 3’SL, averaging

230

an excess of 29% abundance across all time points (Figure 2B). 3’SL was, in fact, the most

231

abundant BMO detected in accordance with previous studies.11,12,19,27 However, with few

232

exceptions such as BMO3 and BMO23 (Figure 2B), no significant differences were observed

233

between the relative levels of BMOs between the grass and grain/corn diet groups (Figure S4),

234

although the divergence amongst pooled samples of raw milk from three different farms, and

235

processed milks from local grocery stores (Table S4) suggests that some environmental influence

236

on BMO composition exists. Nevertheless, the stage of lactation was clearly the dominant source

237

of variation in relative BMO levels with several significantly increasing (BMO 3, 4, 5, 20 and

238

23) to the decrement of others (BMO 12 and 13; Figure 4). Decreases in BMO13 (3’SL) and

239

BMO12, the latter containing Neu5Ac/Neu5Gc as demonstrated by its sensitivity to

240

neuraminidase, indicate that the decrease in absolute Neu5Ac/Neu5Gc levels observed over the

241

first week of lactation (Figure 1D) must be at least partially due to alterations in BMO

242

biosynthetic pathways. In summary, our data do not support the hypothesis that the bovine diet

243

affects BMO structures, although notable differences in the pooled samples from both processed


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and non-processed sources suggest that a larger sample size may permit a more definitive

245

conclusion.

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Characterization of Unexpectedly Acidic BMOs. The significant decrease observed in 3’SL

247

(BMO13) levels, which dropped from a median level of 59% relative abundance to 29% during

248

the first week of lactation, mirrored a nearly identical (2 to 24%), and also significant, increase in

249

BMO3—which was the second most abundant BMO observed in mature milk—over the same

250

time span, thus making the identification of this compound imperative. Also intriguing, was the

251

observation that all BMOs from 1-10 possess unexpectedly high ionic mobilities (Figure 2B).

252

Note that in CE, analyte mobility is dictated by both size (i.e., hydrodynamic radius) and charge,

253

with the larger, neutral BMOs eluting later than the smaller, more anionic ones eluting earlier

254

under the reversed polarity and zero electroosmotic flow CE conditions used here. Thus, for

255

example, attaching a single α-1,3-Gal residue to lactose (BMO14) creates a later eluting

256

trisaccharide (BMO18) while linking a Neu5Ac residue to lactose, which also generates a

257

trisaccharide (3’SL; BMO13), results in a faster migrating species than lactose itself, due to the

258

increased negative charge imparted by the Neu5Ac moiety. Thus, BMOs 1-10 represent species

259

that are apparently either smaller or more highly charged than 3’SL, prompting a more detailed

260

investigation of these structures initially based on their sensitivity (or resistance) to both

261

enzymatic or controlled chemical hydrolysis (Figure 4). It was reasoned that APTS-labelled

262

BMOs would be suitable substrates for neuraminidase or weak acid hydrolysis, both of which

263

would only cleave Neu5Ac or Neu5Gc residues (Figure 4A). Four Neu5Ac-containing BMO

264

standards indeed proved to be neuraminidase substrates, being completely hydrolyzed to either

265

lactose or lactosamine, both of which migrated more slowly than the parent BMOs due to the

266

loss of negative charge (Figure 4B). In contrast, when the BMOs extracted from a colostrum



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sample were subjected to the same conditions, six BMOs (11-13, 17, 21, and 25) were obviously

268

hydrolyzed, as evidenced by their complete loss from the electropherogram produced from the

269

neuraminidase-treated sample (Figure 4C), while the levels of BMOs 1-10 remained essentially

270

unchanged (only BMOs 7-10 are shown here; BMOs 1 – 11 are depicted in Figure S6). Note that

271

under these hydrolysis conditions, no detectible lactosamine was produced indicating that in

272

these samples the lactosamine-based BMOs 3’ and 6’SLN only made minor contributions to the

273

total BMO pool.

274

Neuraminidases are known to be sensitive to both the regiochemistry of the Neu5Ac

275

glycosidic bond (α2,3 vs. 2,6 vs. 2,8) in addition to modifications to the Neu5Ac moiety (i.e., O-

276

acetylation) or the nature of the underlying glycan substrate.34–36 To account for the possibility

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that some Neu5Ac-bearing BMOs could be resistant to neuraminidases, a BMO sample was

278

subjected to weak acid hydrolysis, under conditions which we30 and others37,38 have shown to be

279

sufficient to cleave acid-labile Neu5Ac/Neu5Gc glycosidic bonds. Under these conditions all

280

neuraminidase-labile BMOs previously identified were completely eliminated from the resulting

281

electropherogram, while BMOs 1-4, and 6 remained resistant (Figure 4D). Careful inspection of

282

the data indicated that BMO5 was hydrolyzed, apparently yielding BMO6 which increased

283

considerably in the acid-treated sample. The relative order of migration (BMO5 preceding

284

BMO6) is consistent with BMO5 losing negative charge (i.e., an acidic sugar residue). The slight

285

increase in BMO3 relative to, for example BMO4, may indicate that the former was also

286

produced under the acidic conditions, although in this instance the precursor BMO is less clear.

287

The resistance of BMO7-10 to acid hydrolysis was less obvious (Figure S6) as several new peaks

288

appeared in this region after hydrolysis preventing clear peak assignments.


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Since some of the highly mobile BMOs (1-4, and 6) appeared to be resistant to both

290

neuraminidase and weak acid hydrolysis, we hypothesized that the cause of the increased anionic

291

character (relative to the known Neu5Ac-containing BMOs) was due to the presence of

292

phosphate or sulfate moieties, both of which are smaller than Neu5Ac yet possessing, at the pH

293

of the CE buffer, an equivalent negative charge. Note that several phosphorylated lactose11 and

294

lactosamine analogues,11,39 including a Neu5Ac-bearing phospho-lactosamine,40 have been

295

previously reported. To test for the presence of phosphorylated species, an APTS-labelled BMO

296

sample was treated with alkaline phosphatase (Figure 4E and Figure S7). Under these conditions

297

a single peak, BMO2, appeared to be obviously hydrolyzed; since BMO2 was resistant to both

298

acid-hydrolysis and neuraminidase it can be concluded that this was likely a phospho-

299

lactosamine or phosphor-lactose analogue lacking a Neu5Ac residue. It was reasoned that the

300

significantly increasing levels of BMO3, apparently at the expense of 3’SL (BMO13; Figure 3),

301

suggested that the former peak corresponded to 3’-sulfo-lactose, which would be consistent with

302

this peak’s resistance to phosphatase, neuraminidase and acid-catalyzed hydrolysis and also

303

account for their biosynthetic competition. 3’-Sulfo-lactose, which was commercially-available,

304

was indeed observed to co-migrate with BMO3. To the best of our knowledge, the presence of

305

3’-sulfo-lactose in bovine milk has never previously been reported (although it has been

306

observed in canine milk41 and larger, sulfated HMOs42 have also been characterized) which is

307

striking in light of the fact that after one week of lactation it was the second most abundant

308

BMO, accounting for nearly a quarter of the total relative levels (Figure 2B and Table S3), and

309

was also detected at high levels in pooled samples of fresh and store-bought bovine milk (Table

310

S4).



Page 16 of 38

311

Characterization of sulfated and phosphorylated BMOs by mass spectrometry.

312

Electropherograms of APTS-labelled BMOs indicated the presence of at least 10 BMOs with

313

mobilities exceeding that of 6’SL (BMO11), and with resistance to chemical and neuraminidase-

314

catalyzed hydrolysis, suggesting that these were phosyphorylated and/or sulfated species, a

315

hypothesis proven in part by sensitivity to alkaline phosphatase (BMO2) and co-migration with a

316

standard (BMO3; 3’-sulfo-lactose). HPLC-MS was therefore used to further assess the extracted

317

BMO samples for the presence of phosphorylated and/or sulfated analogues (Figure 5 and Table

318

1). Consistent with the CE data, abundant amounts of 3’-sulfo-lactose (Figure 5, peak g) were

319

observed, matching the accurate mass, retention time and collision induced dissociation (CID)

320

spectrum of an authentic standard (Figure S5). Two phosphorylated disaccharides analogues,

321

composed of an N-acetylhexosamine and a hexose residue, (peaks a and e) were also detected,

322

one of which is likely 6-phospho-lactosamine—since 3’sialyl-6-phospho-lactosamine, although

323

not detected here, has previously been reported (with the phosphate borne on the reducing

324

GlcNAc). The other phosphylated disaccharide is hypothesized to be either 3’ or 6’-phospho-

325

lactosamine.40 In addition, masses consistent with eight sulfated BMOs were detected by HPLC-

326

MS, all of which contained a lactose moiety at the reducing end. These sulfated BMOs included

327

a disaccharide hypothesized to be 6’-sulfo-lactose (peak d), three different trisaccharides (peaks

328

h – j), two tetrasaccharides, one Neu5Ac-containing trisaccharide (peak f), hypothesized to be 6’-

329

sulfo-3’-sialyllactose, and a single trisaccharide bearing two sulfate groups (peak c). The MS/

330

MS spectra of both 3’-sulfo-lactose (peak g) and the putative 6’-sulfo-lactose (peak d) contained

331

a prominent peak at m/z = 241.0023, consistent with the neutral loss of the reducing end Glc

332

residue (Figure S5A-B). An identical fragment was observed for trisaccharide (i), indicating that

333

this compound also contained the sulfate on the non-reducing (rather than internal) hexose.


Page 17 of 38


334

Alternatively, trisaccharide (j) appeared to fragment via the neutral loss of the reducing end Glc

335

moiety, suggesting the presence of a sulfate on the internal Gal residue. Not detected by MS

336

were any compounds consistent with sulfo-lactosamine or phospho-lactose analogues.

337

Sulfated oligosaccharides have been abundantly observed across multiple tissue types, for

338

example in glycosaminoglycans such as heparin sulfate, and glycoproteins like the epithelial

339

cell-expressed sulfomucins43 (reviewed in reference

340

adhesion ligands like GlyCAM-1.45,46 Intriguingly, the latter two classes of glycoproteins have

341

each been demonstrated to bear both 3’- and 6’-sulfo-galactose residues, analogous to those

342

borne by BMOs like 3’- and the putative 6’-sulfo-lactose (Figure 5). GlyCAM-1 has also been

343

demonstrated to be decorated with glycans terminating in 6’-sulfo-sialyl-LewisX epitopes

344

wherein the same non-reducing galactose residue is elaborated with both Neu5Ac (at the 3’ OH)

345

and sulfate (at the 6’ OH) moieties;45 indeed, the only difference between this epitope and the

346

sulfated/sialylated BMO described herein (Figure 5, peak f) would be the substitution of a

347

reducing GlcNAc for the Glc moiety of the BMO. The existence of (at least) eight sulfated

348

BMOs, comprising a major fraction of the total pool, is likely significant to human health for

349

several reasons. First, in humans, the highly glycosylated mucus glycoproteins (i.e., sulfomucins)

350

protecting the gastrointestinal (GI) tract exhibit a gradient of increasing oligosaccharide

351

sulfation, with the highest levels observed in the large intestine and colon, locations in which

352

BMOs/HMOs are thought to exert their primary influence.1–4 Sulfatase-catalyzed uncapping of

353

sulfated GI mucins is considered to be the rate-limiting step in the subsequent degradation of

354

mucus-linked oligosaccharides by GI microbes,44 and such a loss of sulfation on colonic mucins

355

has been correlated with inflammatory GI disorders such as ulcerative colitis.36,47 Thus, the

356

exogenous supply of sulfated BMOs may limit the degradation of host tissues during periods of

44

) and endothelially-expressed cell-



Page 18 of 38

357

GI microbial dysbiosis. Interestingly, Bifidobacterium breve,48 and B. bifidum,49 and Bacteroides

358

thetaiotaomicron,50 all prominent human GI microbes, have each been recently observed to

359

possess sulfatase encoding genes permitting them to metabolize sulfated monosaccharides.

360

Therefore, it is very probable that in addition to serving as pathogen decoys, sulfated BMOs

361

exert specific prebiotic functions in the GI tract. Finally, sulfate-containing glycans are known to

362

be ligands for lectins that play key roles in modulating the immune system. For example, 6’-

363

sulfo-lactose has been demonstrated to be an antagonist of L-selectin46 and thus may directly

364

interfere with leukocyte recruitment to the GI tract. Alternatively, 3’-sulfo-lactose has shown to

365

be recognized and bound by galectin-4 which is predominantly expressed in the GI tract where it,

366

paradoxically, may both exacerbate and/or reduce mucosal inflammation.51 Similarly, 3’-sulfo-

367

lactose has also been shown to bind to P-selectin when presented in the context of the

368

glycolipids,52 and thus may also function as a P-selectin antagonist

369

Interrelationship between BMO concentrations. Spearman rank correlations, which have

370

previously been employed to deduce the directionality and strength of the biosynthetic

371

relationships between HMOs,22,53 were likewise calculated here for BMOs (Figure 6). Recently,

372

a similar correlative analysis on the seasonal influences on BMO concentrations was reported by

373

Liu and co-workers,21 albeit with several notable differences from the approach used here.

374

Specifically, Liu et al. sampled milk from 15 cows (at eight time points over eight months),

375

following the relative abundance of 14 BMOs; the correlation analysis here was performed using

376

six cows (at three time points over one week) but it included 33 BMOs, nine which were

377

identified with standards (Table S3). Several clear correlations between the Neu5Ac and Neu5Gc

378

levels (Figure 1D) and the relative abundances of specific BMOs validated the use of this

379

correlative approach. For example, Neu5Ac and Neu5Gc levels were positively correlated (ρ =


Page 19 of 38


380

0.664) with each other, consistent with Neu5Gc being directly biosynthesized from Neu5Ac

381

(Figure S2). Similarly, the major neuraminidase-sensitive BMOs (12 and 13) also were

382

positively correlated with total Neu5Ac/Neu5Gc levels (ρ >0.65 in all four cases); based on this

383

evidence it is proposed that BMO12 corresponds to the Neu5Gc-analogue of 3’SL, this peak

384

being clearly lactose-derived but not co-migrating with any Neu5Ac-containing standard (Figure

385

4B and 4C). Interestingly, BMO11 (6’SL) was neither correlated with total Neu5Ac levels nor

386

3’SL as might be predicted if there was competition between different Neu5Ac transferases (e.g.,

387

α2,3 vs. α2,6) for a limited pool of the donor monosaccharide cytidine-5’-monophosphate

388

(CMP)-Neu5Ac. Neuraminidase-sensitive BMO17 exhibited a similar lack of correlation

389

between relative abundance and total Neu5Ac/Neu5Gc levels. These data suggest that

390

Neu5Ac/Neu5Gc donors were not supply-limited, at least during the first week of lactation, and

391

that any correlations detected between the Neu5Ac/Neu5Gc-containing BMOs and others, such

392

as the sulfated species, were likely due to changes in the expression levels of other glycosyl- or

393

sulfo-transferases, or enzymes involved in the activities of these. Consistent with this, is the

394

significant negative correlation (ρ = -0.618) that was detected between 3’-sulfo-lactose (BMO3)

395

and 3’-SL (BMO13), a trend that is in agreement with the significant reversal in the relative

396

abundances of these BMOs over the first week of lactation (Figure 3). The correlation analysis

397

revealed that 3’-sulfo-lactose was also significantly correlated with BMO5 (ρ = 0.833), BMO15

398

(ρ = 0.717) and BMO20 (ρ = 0.558), two of which are known to be sulfated: BMO5, based on its

399

electrophoretic mobility, and BMO20 based on the observation that β-galactosidase specifically

400

converted this oligosaccharide to BMO8, a sulfated compound as inferred from its mobility

401

(Table S3 and Figure S8). As observed for 3’-sulfo-lactose, putative sulfated BMOs 5, 15 and 20

402

were all significantly negatively correlated with both total Neu5Ac/Neu5Gc levels and 3’SL, the



Page 20 of 38

403

most abundant Neu5Ac-containing BMO. Based on these correlations, it is hypothesized that

404

BMOs 20 and 15, corresponding to sulfated tetra- and trisaccharides, respectively, are related in

405

that they share a common 3’-sulfo-lactose reducing end. BMO5 is putatively assigned as the

406

sulfate- and Neu5Ac-containing compound identified by MS (Figure 5, peak f), an assignment

407

consistent with its sensitivity to weak acid hydrolysis (Figure 4D) to yield BMO6 which is

408

hypothesized to be 6’-sulfo-lactose. BMO4 is hypothesized to be the disulfated trisaccharide

409

based on its relative electrophoretic mobility and its significant negative correlation to BMOs 8

410

and 10 (ρ = -0.651 and -0.680, respectively) which are likely two of three mono-sulfated

411

trisaccharides, both of which can serve as direct precursors to BMO4. Several experiments are

412

currently in progress in order to unambiguously correlate the sulfated BMOs detected by MS

413

with their corresponding CE peaks.

414

In conclusion, the CE analyses described herein permitted the rapid characterization of

415

the relative abundances of 33 BMOs in milk collected from exclusively grass-fed or grain/corn-

416

fed cows at matched time points during the first week of lactation. Few significant differences

417

were observed between individual animals in these groups, although differences were observed

418

in pooled samples of mature milk. The unexpectedly high electrophoretic mobilities of ten

419

BMOs led us to propose the existence of sulfated species, ten of which were detected by MS and

420

tandem MS. Correlation analyses revealed that the biosynthesis of several sulfated BMOs was in

421

direct competition with that of Neu5Ac/Neu5Gc-containing analogues, 3’-sialyllactose in

422

particular. Although these sulfated BMOs have not been previously described, they are likely to

423

be important to human health given their high relative abundance in mature, processed milk and

424

given that they are close structural analogues to the highly sulfated oligosaccharides lining the

425

human GI tract.


Page 21 of 38


426

Acknowledgments. The authors thank Mike Broersma, Ray Vanderhorst, and Garry and Kathy

427

Wikkerink for graciously collecting and donating the milk samples used in this study.

428

Funding Sources. This research was supported by funding from the Natural Science and

429

Engineering Research Council of Canada (Discovery Grant, 2016-03929). Infrastructure was

430

obtained with the support of the Canada Foundation for Innovation (project number 35246) and

431

the British Columbia Knowledge Development Fund. SDV was supported by scholarships from

432

the British Columbia Proteomics Network and a UBC undergraduate research award; AMG was

433

supported by an NSERC undergraduate student research award (USRA).

434

Supporting Information. The Supporting Information is available free of charge on the ACS

435

publication website. Included are eight figures and four tables.

436

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Raouf, A. H.; Tsai, H. H.; Parker, N.; Hoffman, J.; Walker, R. J.; Rhodes, J. M. sulphation

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of colonic and rectal mucin in inflammatory bowel disease: Reduced sulphation of rectal

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mucus in ulcerative colitis. Clin. Sci. 1992, 83, 623–626.

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Egan, M.; Jiang, H.; Motherway, M. O. C.; Oscarson, S.; Van Sinderen, D.

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Glycosulfatase-encoding gene cluster in Bifidobacterium Breve UCC2003. Appl. Environ.

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Microbiol. 2016, 82, 6611–6623.

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Katoh, T.; Maeshibu, T.; Kikkawa, K. ichi; Gotoh, A.; Tomabechi, Y.; Nakamura, M.;

582

Liao, W. H.; Yamaguchi, M.; Ashida, H.; Yamamoto, K.; et al. Identification and

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Characterization of a sulfoglycosidase from Bifidobacterium bifidum implicated in mucin

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glycan utilization. Biosci. Biotechnol. Biochem. 2017, 81, 2018–2027.

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Cartmell, A.; Lowe, E. C.; Baslé, A.; Firbank, S. J.; Ndeh, D. A.; Murray, H.; Terrapon, N.; Lombard, V.; Henrissat, B.; Turnbull, J. E.; et al. How members of the human gut



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microbiota overcome the sulfation problem posed by glycosaminoglycans. Proc. Natl.

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Acad. Sci. 2017, 114, 7037–7042.

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Bum-Erdene, K.; Leffler, H.; Nilsson, U. J.; Blanchard, H. Structural characterisation of

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human galectin-4 N-terminal carbohydrate recognition domain in complex with glycerol,

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lactose, 3′-sulfo-lactose, and 2′-fucosyllactose. Sci. Rep. 2016, 6, 20289.

592

(52)

binding to sulfatides on tumor cells. Glycobiology 2007, 17, 185–196.

593 594

Garcia, J.; Callewaert, N.; Borsig, L. P-selectin mediates metastatic progression through

(53)

Smilowitz, J. T.; O’Sullivan, A.; Barile, D.; German, J. B.; Lonnerdal, B.; Slupsky, C. M.

595

The human milk metabolome reveals diverse oligosaccharide profiles. J. Nutr. 2013, 143,

596

1709–1718.

597

Figure captions

598

Figure 1. Quantification of total BMO and monosaccharide levels in milk from corn/grain (c/g)-

599

or grass (gr)-fed cows. (A) Total reducing BMO levels. (B) Representative CE electropherogram

600

containing standards and a BMO hydrolysate used for (C) determining the levels of neutral

601

monosaccharides relative to Glc. (D) Absolute amounts of the acidic monosaccharides, Neu5Ac

602

and Neu5Gc. n = 3 biological replicates for the three time points in each diet group; pooled tank

603

samples were extracted and analyzed in triplicate. All data are reported ± 1 standard error of the

604

mean (SEM).

605

Figure 2. Relative BMO levels established by CE with fluorescence detection. (A)

606

Representative CE electropherogram of milk (colostrum) from a grass (only)-fed cow. Peak

607

areas for the first 34 BMOS, in order of migration, were determined for all samples. (B) Relative

608

levels of selected BMOs. Open circles represent each biological replicate; closed squares are


Page 29 of 38


609

mean values with the bars corresponding to the SEM. * indicates a significant difference

610

(Student’s t test; p < 0.05) between the two groups that time point.

611

Figure 3. Relative changes in major BMOs as a function of lactation time. BMOs are numbered

612

as shown if Figure 2. Each box-whisker plot contains six biological replicates, three animals

613

from each farm. Center lines show medians, box limits indicate the 25 and 75 percentiles,

614

whiskers extend to 1.5 times the interquartile range from the 25 and 75 percentiles, points

615

beyond these ranges were assigned as outliers. Statistical differences between groups were

616

assessed using a Mann-Whitney U test (* p < 0.05; ** p < 0.01).

617

Figure 4. Characterization of highly anionic BMOs based on their sensitivity or resistance to

618

weak acid- or enzyme-catalyzed hydrolysis. (A) Both mild acid (acetic acid = AcOH) and

619

neuraminidase were predicted to selectively hydrolyze Neu5Ac/Neu5Gc-bearing, APTS-labelled

620

BMOs which was proven (B) using commercially-available standards. (C) Neuraminidase failed

621

to hydrolyze BMOs of higher mobility than 6’SL (BMO11) but BMO11-13, 17, 21 and 25 were

622

completely digested (D) All neuraminidase-sensitive BMOs were also acid-labile under

623

conditions that failed to obviously hydrolyze BMO1-4, and 6, while relative levels of BMO3 and

624

6 increased. (E) Alkaline phosphatase obviously hydrolyzed BMO2. Note that different milk

625

samples were used to prepare Figure 2, and 4C- 4E; 2,4C and 4E were colostrum (day 0) samples

626

while 4D was a pooled sample of mature milk.

627

Figure 5. Sulfated and phosphorylated BMOs detected by MS. Extracted ion chromatograms of

628

ten putative BMOs (Table 1). All BMOs reported were from the same HPLC-MS injection. For

629

comparative purposes the intensity of peak g (3’-sulfo-lactose) and peaks h – j have been scaled

630

down 500- and 100-fold, respectively; peak c has been scaled up 10-fold.

th

th

th

th



Page 30 of 38

631

Figure 6. Spearman rank correlation heatmap depicting the relationships between the 33 BMOs

632

detected in all individual milk samples. BMOs are numbed as depicted in Figure 2; structures are

633

indicated only where BMOs have been identified based on their co-migration with commercial

634

standards (see Figure S1 for full names and a list of all standards tested). Color indicates

635

directionality (red = positive; blue = negative) and intensity indicates the magnitude of each

636

association.


Page 31 of 38


Tables Table 1. Retention time (RT) and MS parameters for the HPLC-MS analysis of sulphated and phosphorylated bovine milk oligosaccharides. Peaka

RT (min)

Formula

Compositionb

Exact mass (Da)

δ (ppm)

a

5.0

C14H28NO14P-

PO3 + Hex + HexNAc

464.1160

+ 1.94

b

5.0

C24H42O24S

SO3+ 4Hex

746.1805

- 2.41

c

8.2

C18H34O22S2

2 SO3 + 3Hex

666.0983

-9.18

d

10.6

C12H24O14S

SO3 + 2Hex

424.0925

+ 8.92

e

12.2

C14H28NO14P- PO3 + Hex + HexNAc

464.1192

- 4.96

f

13.0

C23H41NO22S

SO3 + Neu5Ac+2Hex

715.1794

+ 6.57

g

15.0

C12H24O14S

3’-sulfo-lactose

424.0896

- 2.12

h

17.0

C18H34O19S

SO3 + 3Hex

586.1455

- 6.82

i

17.7

C18H34O19S

SO3 + 3Hex

586.1468

- 9.04

j

18.8

C18H34O19S

SO3 + 3Hex

586.1361

+ 9.21

k

22.0

C24H42O24S

SO3 + 4Hex

746.1800

- 1.74

a

Peaks are labelled as in Figure 5. bHex = hexose; HexNAc = N-acetylhexosamine (most likely GlcNAc); SO3 and PO3 = sulfate and phosphate, respectively.



Page 32 of 38

Figure graphics

Figure 1.


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Figure 2.



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Figure 3.


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Figure 4.



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Figure 5


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Figure 6.



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Graphic for table of contents


Capillary Electrophoresis Analysis of Bovine Milk Oligosaccharides

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