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New insights into porcine milk N-glycome and the potential relation with offspring gut microbiome Chunlong Mu, Zhipeng Cai, Gaorui Bian, Yamin Du, Shouqing Ma, Yong Su, Li Liu, Josef Volgmeir, Ruihua Huang, and Weiyun Zhu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00789 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on December 23, 2018

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Journal of Proteome Research 1

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New insights into porcine milk N-glycome and the potential relation with

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offspring gut microbiome

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Chunlong Mu †‡§⊥ , Zhipeng Cai ‖⊥ , Gaorui Bian †‡§ , Yamin Du ‖ , Shouqing Ma †‡§ , Yong

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Su†‡§, Li Liu‖, Josef Volgmeir‖, Ruihua Huang†, Weiyun Zhu*†‡§

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† Laboratory

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‡Jiangsu

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for International Research on Animal Gut Nutrition, ‖Glycomics and Glycan Bioengineering

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Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural

of Gastrointestinal Microbiology, College of Animal Science and Technology,

Key Laboratory of Gastrointestinal Nutrition and Animal Health, §National Center

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University, Nanjing 210095, China

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⊥These

authors contributed equally to this work.

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ACS Paragon Plus Environment

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ABSTRACT: N-glycans are an important source of milk oligosaccharides. In addition to

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free oligosaccharides found in milk, N-glycans can also be utilized by gut microbes. A

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potential for milk N-glycans to act as gut microbe regulators in suckling animals has

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attracted considerable attention; however, sow milk N-glycans and their potential effects

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upon the piglet’s gut microbes in vivo remain unknown. In the present study, we profiled

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the milk N-glycans of Meishan and Yorkshire sows during lactation using UPLC and a mass

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spectrometry-based glycome method, and explored the correlations between milk N-

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glycans and offspring gut microbiota. Twenty-two N-glycan structures were identified in

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sow milk, among which 36% (8 out of 22) were fucosylated, 41% (9 out of 22) were

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sialylated, and 14% (3 out of 22) were high mannosylated. An N-glycan with a NeuGc

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structure was identified in sow milk for the first time. No compositional differences between

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the two breeds or between different lactation times were found in porcine milk N-linked

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oligosaccharides (PNOs); however, the abundances of different structures within this class

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did vary. The relative abundances of fucosylated PNO3 (GlcNAc4-Man3-Fuc) and sialylated

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PNO18 (GlcNAc4-Man3-Gal2-NeuAc) increased during lactation, and Meishan sows

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demonstrated a higher (P < 0.05) abundance of mannosylated PNO10 (GlcNAc2-Man6)

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and sialylated PNO17 (GlcNAc5-Man3-Gal-NeuAc) than Yorkshire sows. Apparent

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correlations between milk N-glycans and offspring gut microbial populations were found;

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for example, mannosylated PNO21 (GlcNAc2-Man9) was positively correlated with OTU706

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(Lactobacillus amylovorus) and OTU1380 (Bacteroides uniformis). Overall, our results

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indicate that the sow milk N-glycome of Meishan and Yorkshire sows differs in N-glycome

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characteristics, and that this is correlated to abundances of certain piglet gut microbes.


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These findings provide a reference for future elucidation of the involvement of gut microbes

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in milk N-glycan metabolism, which is important to the health both of large domestic

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animals and humans.

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KEYWORDS: breed, lactation time, N-glycome, porcine milk N-glycan oligosaccharides



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■ INTRODUCTION

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Milk oligosaccharides are essential nutrients for neonates, and also stimulate the growth

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of beneficial microbes and participate in pathogen inhibition in the gut.1 Milk

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oligosaccharides include both free and conjugated oligosaccharides, and free

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oligosaccharides have already been shown to be utilized by intestinal microbes.1 In

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addition to free oligosaccharides, N-glycans, one of the major conjugated sugars, are also

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an important component of milk oligosaccharides and have demonstrated pathogen-

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inhibiting activity.2 N-glycans are oligosaccharides attached to the asparagine residues of

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a protein via N-acetylglucosamine linkages. The compositions and structures of free

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oligosaccharides in sow milk have been well-elucidated3, 4; however, the N-glycan

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component in sow milk has yet to be explored.

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Breed differences affect milk composition in sows and thus nutrients for neonates. For

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example, Meishan (a typical obese Chinese breed) sows have a higher concentration of

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milk fats and calcium than Large White (Yorkshire, a lean type breed) sows.5, 6 Interestingly,

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Meishan and Yorkshire pigs differ in genes encoding fucosyltransferases7 which mediate

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the transfer of fucose onto N-glycan chains. In a survey of the genotype frequency of the

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porcine fucosyltransferase 1 gene, at the 307 bp loci, Meishan pigs were found to have a

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higher frequency of GG motifs, while Yorkshire pigs presented with both GG and AG

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motifs.7 Glycoprotein profiles in the ileum mucosa of Yorkshire pigs showed that those with

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the AG motif have a lower abundance of sialylated glycans than those with an AA motif.8

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These results suggest potential important differences in glycan patterns among pig breeds.

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However, whether breed affects milk N-glycan composition has yet to be determined.


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Recently, milk glycans have been shown to be an important substrate for gut

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microbiota. Both glycoproteins and their glycan components can be utilized by specific

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microbes. Bifidobacterium infantis use endo-β-N-acetylglucosaminidase to release

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complex N-glycans from human milk glycoproteins.9 Bacteroides fragilis can also

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deglycosylate glycoprotein by producing endo-β-N-acetylglucosaminidase which in turn

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generates glycans that can be used for bacterial growth.10 Bacteroides thetaiotaomicron

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can

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glucosamine phosphorylase for degradation of mannosylated N-glycans.11 The human milk

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N-glycome can promote the growth of Bifidobacterium longum subsp. infantis, which

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utilizes N-glycans with a preference for small structures.12 These results suggest that

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variations among preferred milk glycan substrates by disparate microbial species could

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affect gut microbial composition. In our previous study, we cross-fostered newborn piglets

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between genetically obese Meishan and lean Yorkshire sows, and found that milk

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appeared to affect the fecal microbiota composition of the pre-weaned piglets.13 However,

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little is known about the in vivo effects of sow milk N-glycan composition on gut microbial

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colonization in offspring.

secrete

endo-β-N-acetylhexosaminidase

and

β-1,4-D-mannosyl-N-acetyl-D-

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In the present study, we further analyzed the milk N-glycan compositions of Meishan

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and Yorkshire sows during lactation. We also delineated the microbial population

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succession in piglets to explore potential correlations between the piglet gut microbiota and

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milk N-glycans. The interest of profiling sow milk and the pig microbiome is to provide a

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rational scientific basis for future studies on the impact of glycans on gut microbiota in this

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and other large domestic species. The pattern of early colonization and acquisition of gut



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microbiota of the piglet is similar to that in human infants during breastfeeding in many

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aspects, such as increasing α diversity, gradually increasing Bacteroidetes spp.

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abundance, and decreasing Proteobacteria abundance.13, 14 In the present study, we use

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the piglet as a model to elucidate the relationship between milk N-glycans and offspring

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gut microbiota composition. We hypothesize that milk N-glycans may be related to the

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alteration of early-life gut microbiota colonization in offspring. By profiling the composition

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of milk N-glycans and examining the in vivo correlation of these glycans to gut microbiota,

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we intend to provide the basis for future research into the involvement of specific microbes

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in milk N-glycan metabolism, as well as underscore its importance in animal and human

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health. These data may also provide a reference for regulating milk nutrition and seeking

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strategies for assisting infants subject to milk deficiency.

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■ MATERIALS AND METHODS

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Ethics

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Animals were managed throughout the study in accordance with requirements for the

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Experimental Animal Care and Use guidelines of Chinese Science and Technology

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Committee, 1998.

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Experimental design

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All Meishan and Yorkshire pigs were raised under the same conditions on a commercial

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farm in Jiangsu Province, China. A cross-fostering approach was adopted with a

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randomized complete block design [2 treatments (breed) *2 block (nursing) with 10

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replicates per group] as described previously.13 Briefly, after vaginal delivery, half of the

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piglets in a litter of a Meishan sow were fostered onto a Yorkshire sow before the piglets


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suckled their mothers’ colostrum. The other half of each litter remained with the birth

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mother. In total, 10 sows of each breed with litters of 10 or 12 piglets were used for the

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cross-fostering operation in delivery-matched pairs. The gestational length of all sows was

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113 –114 d. Diet composition was the same for Yorkshire and Meishan sows, as shown in

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Table S1. Finally, four groups of piglets were generated as follows: Meishan piglets

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fostered by their birth mother (Mm), Yorkshire piglets fostered by Meishan sows (My),

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Meishan piglets fostered by Yorkshire sows (Ym), and Yorkshire piglets fostered by their

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birth mother (Yy). From day 14 after birth, all suckling piglets were offered creep feed ad

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libitum and had free access to water.

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Milk samples from the sows of both breeds were collected when the piglets were 1, 3,

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7, 14 and 21 days old (n = 6 for each sows at sample days). The sample replicates were

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chosen in order to balance the cost of replicates and the number of replicates necessary

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for statistical analysis. The fecal samples from offspring piglets of the chosen sows, from

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which milk samples were collected, were used for microbiome analysis. The first fecal and

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milk samples were collected within 12 h after birth, while other samples were collected

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between 7:00 and 9:00 AM on the sampling day. A total of 60 samples were used for glycan

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analysis.

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Glycoprofiling of Porcine Milk N-glycan Oligosaccharides (PNOs)

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N-glycans preparation. The stored frozen milk samples were completely thawed to room

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temperature and protein was subsequently precipitated using chloroform and methanol.15

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The N-glycans were obtained by N-glycanase F (PNGase F) digestion followed by solid-



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phase extraction with non-porous graphitized carbon cartridges to remove the buffer salts

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prior to 2-AB labelling.

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aminobenzamide (2-AB) prior to ultra performance liquid chromatography (UPLC) and

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exoglycosidase assays.17

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Then, the purified N-glycans were fluorescently labeled with 2-

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Hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC) for

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profiling of milk N-glycome. 2-AB-labeled N-glycan samples were profiled on an Acquity

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UPLC BEH Glycan Column (2.1×150 mm, 1.7 µm particle size; Waters, Ireland) at a

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column temperature of 60°C, using the Nexera UPLC system (Shimadzu Corporation,

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Kyoto, Japan) consisting of a LC-30AT pump system and a RF-20Axs fluorescence

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detector (excitation 330 nm, emission 420 nm). Solvent A and B were aqueous ammonium

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formate buffer (50 mM, pH 4.5) and acetonitrile respectively. The separation was

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performed using a linear gradient of 5–12% of solvent A from 0-6 min, solvent B was then

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increased to 44.1% over 39 min followed by a further increase to 100% over 3 min and

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held at 100% for 1 min. The solvent A (aqueous ammonium formate buffer, 50 mM, pH

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4.5) was then decreased to 5% in 7 min and the column was equilibrated with the initial

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conditions for 7 min. The flow rate was 0.5 ml/min from 0-44.5 min, reduced to 0.25 ml/min

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from 44.5-55 min and increased back to 0.5 ml/min from 55-57 min. Different flow rates

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wee used to reduce the backpressure of BEH Glycan Column. The method of

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chromatographic separation is based on the manual of BEH Glycan Column (2.1×150 mm,

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1.7 μm particle size). The optimal flow rate for the elution of N-glycans is 0.5mL/min. After

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that, it is necessary to wash the column several minutes in 100% solvent A (aqueous

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ammonium formate buffer, 50 mM, pH 4.5) before next sample. However, the backpressure


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will be very high in this condition. Therefore, we reduced the flow rate to 0.25mL/min during

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the chromatographic separation. This procedure can benefit the column by avoiding the

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high pressure and maximizing the lifetime of column and system. The column was

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calibrated with a 2AB-labeled dextran standard (2-20 glucose units), and the absolute

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retention time of each N-glycan peak was converted to relative retention units based on

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the dextran-ladder-derived glucose units (GU value) to avoid experimental error. UPLC

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fractions with selected peaks were manually collected for mass spectrometry assay.

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Exoglycosidase assay. In order to elucidate the glycan structure, N-glycans were

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subjected to exoglycosidase assays. All the enzymes employed in this study were obtained

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from Prozyme (San Leandro, CA, USA) and used according to the manufacturer’s

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instruction. After Jack bean α-mannosidase digestion, the sample was divided into five

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parts. One part was directly analyzed by HILIC-UPLC, whereas the others were subjected

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to further sequential glycosidase treatment including: sialidase A, bovine kidney α-

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fucosidase, bovine testis β-galactosidase, alpha-galactosidase, and jack bean β-N-

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acetylhexosaminidase. Samples were then analyzed by UPLC.

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Mass Spectrometry (MS). 2AB-labelled N-glycans from UPLC were analyzed by

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matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Autoflex;

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Bruker Daltonics, Bremen, Germany). The MS spectra were acquired in monoisotopic

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resolution using a Bruker Autoflex Speed (equipped with a 2000 Hz Smartbeam™‐II laser)

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instrument (Bruker Daltonics, Bremen, Germany). 6-aza-2-thiothymine was used as the

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matrix (3 mg/ml in 70% aqueous acetonitrile solution). MS/MS was performed by laser-

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induced dissociation. Spectra were processed with the manufacturer’s software (Bruker



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Flex Analysis software version 3.3.80) using the SNAP algorithm with a signal/noise

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threshold of 6 for MS (unsmoothed) and 3 for MS/MS (four times smoothed). The MS and

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MS/MS spectra were interpreted using GlycoWorkbench version 1.1 (database: CFG,

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Carbbank and Glycosciences),18 with the attached labeling reagent 2AB being taken into

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account. Permethylation of N-glycan was carried out according to the method of Harvey

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and coworkers to protect the sialylated glycans from the desialylation during the MS

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detection.19

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Analysis of 16S rRNA pyrosequencing data

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In our previous study,13 fecal samples were collected from piglets at day 1, 3, 7, and 14 of

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age. The total genomic DNA was isolated from fecal samples by the bead-beating method

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as described by Zoetendal and colleagues.20 Amplicon pyrosequencing was performed

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from the A-end using a 454/Roche A sequencing primer kit on a Roche Genome

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Sequencer GS-FLX Titanium platform. The processes of amplification, amplicon

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purification, check of amplicon quantity and quality have been described previously.13 The

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microbial diversity and composition at the phylum and genus level have been reported

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previously.13 To link PNOs with fecal bacteria species, we further analyzed the operational

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taxonomic units (OTUs) using the microbiota dataset.

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The sequence filtering and chimeric trimming were conduced using QIIME software

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package.21 Using the average neighbor algorithm with a cutoff of 97% similarity, sequences

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were clustered to OTUs. Representative sequences from each OTU were taxonomically

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classified with a 90% confidence level using Ribosomal Database Project classifier.22 The

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heatmap of microbial composition was visualized using the Heatmap Illustrator version


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1.0.23

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Statistical analysis

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The data of porcine milk N-glycan oligosaccharides at different time points were analyzed

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using two-way repeated analysis of variance procedures as implemented in MetaboAnalyst

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3.0.24 Comparisons of N-glycans at each time point were conducted using Student’s t test

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(SPSS version 20.0; SPSS Inc., Chicago, Illinois). P-values < 0.05 were regarded as

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statistically significant. The relative abundances of bacterial taxa at the OTU level were

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analyzed using the general lineal mode procedures as implemented in SPSS (SPSS

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version 20.0; SPSS Inc., Chicago, Illinois). In case of multiple comparisons, P-values were

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adjusted with Benjamini-Hochberg multiple testing correction, limiting the overall false

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discovery rate to 5% (q < 0.05). 0.05< q

New Insights into Porcine Milk - ACS Publications - American

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