Milk Glycans Research Articles

2'-Fucosyllactose Inhibits Human Norovirus Replication in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis worldwide. Currently, there are no targeted antivirals for the treatment of HuNoV infection. Histo-blood group antigens (HBGAs) on the intestinal epithelium are cellular attachment factors for HuNoVs; molecules that block the binding of HuNoVs to HBGAs thus have the potential to be developed as antivirals. Human milk oligosaccharides (HMOs) are glycans in human milk with structures analogous to HBGAs. HMOs have been shown to act as decoy receptors to prevent the attachment of multiple enteric pathogens to host cells. Previous X-ray crystallography studies have demonstrated the binding of HMO 2'-fucosyllactose (2'FL) in the same pocket as HBGAs for some HuNoV strains. We evaluated the effect of 2'FL on the replication of a globally dominant GII.4 Sydney [P16] HuNoV strain using human intestinal enteroids (HIEs) from adults and children. A significant reduction in GII.4 Sydney [P16] replication was seen in duodenal and jejunal HIEs from multiple adult donors, all segments of the small intestine from an adult organ donor and in two pediatric duodenal HIEs. However, 2'FL did not inhibit HuNoV replication in two infant jejunal HIEs that had significantly lower expression of α1-2-fucosylated glycans. 2'FL can be synthesized in large scale, and safety and tolerance have been assessed previously. Our data suggest that 2'FL has the potential to be developed as a therapeutic for HuNoV gastroenteritis.

Core fucosylation of maternal milk N-glycans imparts early-life immune tolerance through gut microbiota-dependent regulation in RORγt+ Treg cells.

Milk glycans play key roles in shaping and maintaining a healthy infant gut microbiota. Core fucosylation catalyzed by fucosyltransferase (Fut8) is the major glycosylation pattern on human milk N-glycan, which was crucial for promoting the colonization and dominant growth of Bifidobacterium and Lactobacillus spp. in neonates. However, the influence of core-fucose in breast milk on the establishment of early-life immune tolerance remains poorly characterized. In this study, we found that the deficiency of core-fucose in the milk of maternal mice caused by Fut8 gene heterozygosity (Fut8+/-) resulted in poor immune tolerance towards the ovalbumin (OVA) challenge, accompanied by a reduced proportion of intestinal RORγt+ Treg cells and the abundance of Lactobacillus spp., especially L. reuteri and L. johnsonii, in their breast-fed neonates. The administration of the L. reuteri and L. johnsonii mixture to neonatal mice compromised the OVA-induced allergy and up-regulated the intestinal RORγt+ Treg cell proportions. However, Lactobacillus mixture supplementation did not alleviate allergic responses in RORγt+ Treg cell-deficient mice caused by Rorc gene heterozygosity (Rorc+/-) post OVA challenge, indicating that the intervention effects depend on the RORγt+ Treg cells. Interestingly, instead of L. reuteri and L. johnsonii, we found that the relative abundance of another Lactobacillus spp., L. murinus, in the gut of the offspring mice was significantly promoted by intervention, which showed enhancing effects on the proliferation of splenic and intestinal RORγt+ Treg cells in in vitro studies. The above results indicate that core fucosylation of breast milk N-glycans is beneficial for the establishment of RORγt+ Treg cell mediated early-life immune tolerance through the manipulation of symbiotic bacteria in mice.

Factors Associated with Exclusive Breastfeeding at the Bulili Health Center, South Bureaubuli Village, Palu City

Exclusive breastfeeding is the best nutrient for babies. The impacts of not receiving exclusive breastfeeding are infection, high risk of infectious diseases such as diarrhea and pneumonia, low immune system, and decreased brain development. breastfeeding reduces baby and toddler morbidity and mortality. Human milk glycans, which contain oligosaccharides in both their free and conjugated forms, are demonstrated in studies to be a component of a natural immune process that explains how human milk protects breastfed children from diarrheal illness. This study aims to analyze factors associated with exclusive breastfeeding in the work area of Bulili Public Health Center South Birobuli Subdistrict Palu. This quantitative research was conducted through a cross-sectional approach. This study involved 90 mothers with babies aged 7-12 months, which was carried out from March 2023. The sample was selected using simple random sampling based on inclusion criteria. Direct interviews were used to collect primary data, and secondary data were obtained from Indonesia's health profile, data from the Palu City Health Office 2021, and data on exclusive breastfeeding. The data were processed using the chi-square statistical test with a significance degree of α = 0.05. The results of the study were knowledge (p=0.018), attitude (p=0.001), interest in formula milk promotion (p=0.018), and family support (p=0,002). In Conclusion, there is a significant association between knowledge, attitude, interest in formula milk promotion, and family support with exclusive breastfeeding practice. It is suggested for mothers to increase knowledge, attitude, and family support by gaining information and using social media appropriately.

Human Milk Oligosaccharides And Site-Specific Glycosylation Heterogeneity Of Filipino Human Milk Glycoproteins Exhibit Dynamic Variations During Lactation As Influenced By Secretor Status And Coastal Environmental Condition – A Preliminary Study

Human Milk Oligosaccharides (HMOs) have a role in the immune system development of infants. HMOs and the N-glycans of human milk glycoproteins, especially highly fucosylated ones, function as prebiotics to the bifidogenic gut microbiota. This study reports the first longitudinal profile of HMOs and human milk glycoproteins from coastal, noncoastal, secretor and nonsecretor Filipino mothers analyzed and annotated using QTOF LC/MS and QqQ LC/MS - MRM, respectively. A milk glycan profile from coastal mothers revealed higher (P<0.05) presence of fucosylated branched HMOs, such as DFLNHa, LDFT, LNDFH I, and LNFP II. Moreover, secretor mothers expressed the highest 2’FL from colostrum (Day 5) until the end of the study (Month 6). Interestingly, the predominance of secretory IgA (64%) on the total milk glycoproteins from Filipino mothers supported their biological adaptation in conferring immune protection to their breastfeeding infant. Glycoform variations on milk glycoproteins, sIgA, IgG, Lactoferrin, and a1-Antitrypsin, in coastal mothers were observed. Moreover, lactoferrin was found to be highly glycosylated with 20 fucosylated and sialylated glycoforms. These results suggest that the coastal mother’s unique long-term seafood diet may have influenced their milk glycan profile and the potential incorporation of L-fucose derived from seafood in the biosynthesis of their milk glycans.

Associations among Milk Microbiota, Milk Fatty Acids, Milk Glycans, and Inflammation from Lactating Holstein Cows.

Milk oligosaccharides (MOs) can be prebiotic and antiadhesive, while fatty acids (MFAs) can be antimicrobial. Both have been associated with milk microbes or mammary gland inflammation in humans. Relationships between these milk components and milk microbes or inflammation have not been determined for cows and could help elucidate a novel approach for the dairy industry to promote desired milk microbial composition for improvement of milk quality and reduction of milk waste. We aimed to determine relationships among milk microbiota, MFAs, MOs, lactose, and somatic cell counts (SCC) from Holstein cows, using our previously published data. Raw milk samples were collected at three time points, ranging from early to late lactation. Data were analyzed using linear mixed-effects modeling and repeated-measures correlation. Unsaturated MFA and short-chain MFA had mostly negative relationships with potentially pathogenic genera, including Corynebacterium, Pseudomonas, and an unknown Enterobacteriaceae genus but numerous positive relationships with symbionts Bifidobacterium and Bacteroides. Conversely, many MOs were positively correlated with potentially pathogenic genera (e.g., Corynebacterium, Enterococcus, and Pseudomonas), and numerous MOs were negatively correlated with the symbiont Bifidobacterium. The neutral, nonfucosylated MO composed of eight hexoses had a positive relationship with SCC, while lactose had a negative relationship with SCC. One interpretation of these trends might be that in milk, MFAs disrupt primarily pathogenic bacterial cells, causing a relative increase in abundance of beneficial microbial taxa, while MOs respond to and act on pathogenic taxa primarily through antiadhesive methods. Further research is needed to confirm the potential mechanisms driving these correlations. IMPORTANCE Bovine milk can harbor microbes that cause mastitis, milk spoilage, and foodborne illness. Fatty acids found in milk can be antimicrobial and milk oligosaccharides can have antiadhesive, prebiotic, and immune-modulatory effects. Relationships among milk microbes, fatty acids, oligosaccharides, and inflammation have been reported for humans. To our knowledge, associations among the milk microbial composition, fatty acids, oligosaccharides, and lactose have not been reported for healthy lactating cows. Identifying these potential relationships in bovine milk will inform future efforts to characterize direct and indirect interactions of the milk components with the milk microbiota. Since many milk components are associated with herd management practices, determining if these milk components impact milk microbes may provide valuable information for dairy cow management and breeding practices aimed at minimizing harmful and spoilage-causing microbes in raw milk.

Human milk whey glycoprotein N-glycans varied greatly among different maternal secretor status

Human milk glycans are complex carbohydrates, which play a pivotal role in infant health and neonatal development. Maternal secretor status is known to affect free oligosaccharides in milk. Here, the milk N-glycome of secretor (Se+) and nonsecretor (Se−) individuals was qualitatively and quantitatively analyzed by hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometry. The total glycosylation, fucosylation, and sialylation of N-glycans was three times higher in the Se+ group compared to the Se− group (p < 0.001) per equal volume of milk. Importantly, 52 out of 63 N-glycans—including the eight most abundant ones—differed greatly between Se+ and Se− individuals (p < 0.05). Moreover, nine N-glycans (H5N3F1, H6N3, H3N5F1, H5N5F1, H5N5F1S1, H5N4F3S1, H6N4F2S1, H6N5F4S1, and H8N7S1) were >10 times more abundant in Se+ milk than in Se− milk. These findings lay a glycomics-basis for designing personalized nutrition supplements for infants.

Enzymatic production of a suite of human milk oligosaccharides directly in milk

Human milk oligosaccharides (HMOs) denote specific glycans in human breast milk. They function as prebiotics, immune modulating, and antimicrobial agents in the gut of breastfed infants, and certain HMOs even promote the cognitive development of the baby. HMOs are virtually absent in cow’s milk and hence in infant formula, which provides a huge incentive for identifying ways in which HMOs can be produced to improve infant formulas. Here, we show that different sialylated and fucosylated HMOs can be generated in cow’s milk via different simultaneous enzymatic transglycosylation reactions catalyzed by an engineered sialidase (EC 3.2.1.18, from Trypanosoma rangeli) and an 1,2-α-L-fucosidase (EC 3.2.1.63, from Tannerella forsinthia) acting on the lactose in the milk and on casein glycomacropeptide, two types of commercially available HMOs, i.e. 2′-fucosyllactose and lacto-N-neotetraose, added to the milk. We also outline the details of the individual reactions in aqueous systems, demonstrate that the enzymatic reactions can be accomplished at 5 °C, and validate the products formed by LC-MS and NMR analysis. Enzymatic production of HMOs directly in milk provides opportunities for enriching milk and infant formulas and extends the use of enzymatic transglycosylation reactions to synthesis of HMOs in milk and eventually in other beverages.

Variations in Oligosaccharides and N/O-Glycans in Human Milk through the Eight-Month Lactation Period.

Oligosaccharides and N/O-glycans are abundant in human milk and have numerous biological functions (for instance sialylated glycans provide sialic acid for the growth of infant brains), but their variation trends during lactation need further exploration. Qualitative and quantitative analyses of oligosaccharides and N/O-glycans in human milk at different lactation stages (from 7 days to 8 months) were performed using UHPLC-ESI-MS/MS. Thirty-four oligosaccharides, twenty-three N-glycans, and six O-glycans were identified. Oligosaccharides showed the highest abundance in human colostrum and decreased with the progression of lactation, and the abundance of N/O-glycans fluctuated as lactation progressed, while a high abundance of sialylated oligosaccharides and sialylated N/O-glycans was observed in human colostrum. These findings provide evidence for breastfeeding support and contribute to the development of infant formula supplemented with human milk glycans.

Strategies for Enhancing Microbial Production of 2′-Fucosyllactose, the Most Abundant Human Milk Oligosaccharide

Human milk oligosaccharides (HMOs), a group of structurally diverse unconjugated glycans in breast milk, act as important prebiotics and have plenty of unique health effects for growing infants. 2'-Fucosyllactose (2'-FL) is the most abundant HMO, accounting for approximately 30%, among approximately 200 identified HMOs with different structures. 2'-FL can be enzymatically produced by α1,2-fucosyltransferase, using GDP-l-fucose as donor and lactose as acceptor. Metabolic engineering strategies have been widely used for enhancement of GDP-l-fucose supply and microbial production of 2'-FL with high productivity. GDP-l-fucose supply can be enhanced by two main pathways, including de novo and salvage pathways. 2'-FL-producing α1,2-fucosyltransferases have widely been identified from various microorganisms. Metabolic pathways for 2'-FL synthesis can be basically constructed by enhancing GDP-l-fucose supply and introducing α1,2-fucosyltransferase. Various strategies have been attempted to enhance 2'-FL production, such as acceptor enhancement, donor enhancement, and improvement of the functional expression of α1,2-fucosyltransferase. In this review, current progress in GDP-l-fucose synthesis and bacterial α1,2-fucosyltransferases is described in detail, various metabolic engineering strategies for enhancing 2'-FL production are comprehensively reviewed, and future research focuses in biotechnological production of 2'-FL are suggested.

Nutritional strategies for mucosal health: the interplay between microbes and mucin glycans.

Many aspects of the mechanisms underlying the symbiosis between humans and gut microbes remain unknown and encompass some of the most intriguing questions in microbiome research. An important factor in this symbiosis is the interplay between microbes and human-produced glycans in mucin and breast milk. In this Opinion paper, I propose a synergy between the structural diversity of human mucin glycans and the enzymatic repertoire of the gut microbiome. The contribution of microbes to mucosal health is discussed, and the role of breast milk glycans in mucosal colonization by microbes is explained. The use of prebiotic mucin glycans in general, and specialized infant and medical nutrition in particular, should be considered as the field of interest to modulate the microbiota and improve mucosal health.

Naturally Occurring Glycosidases in Milk from Native Cattle Breeds: Activity and Consequences on Free and Protein Bound-Glycans.

Little is known about the extent of variation and activity of naturally occurring milk glycosidases and their potential to degrade milk glycans. A multi-omics approach was used to investigate the relationship between glycosidases and important bioactive compounds such as free oligosaccharides and O-linked glycans in bovine milk. Using 4-methylumbelliferone (4-MU) assays activities of eight indigenous glycosidases were determined, and by mass spectrometry and 1H NMR spectroscopy various substrates and metabolite products were quantified in a subset of milk samples from eight native North European cattle breeds. The results showed a clear variation in glycosidase activities among the native breeds. Interestingly, negative correlations between some glycosidases including β-galactosidase, N-acetyl-β-d-glucosaminidase, certain oligosaccharide isomers as well as O-linked glycans of κ-casein were revealed. Further, a positive correlation was found for free fucose content and α-fucosidase activity (r = 0.37, p-value < 0.001) indicating cleavage of fucosylated glycans in milk at room temperature. The results obtained suggest that milk glycosidases might partially degrade valuable glycans, which would result in lower recovery of glycans and thus represent a loss for the dairy ingredients industry if these activities are pronounced.

Association between udder inflammation and glycosidase activities and free sugar levels in bovine milk

Two milk glycosidases [N-acetyl-β-glucosaminidase (NAGase) and β-glucuronidase] have previously been associated with elevated somatic cell count (SCC). However, several other glycosidases are present in milk, potentially having effects on milk glycans. The activities of eight glycosidases (NAGase, α-fucosidase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, α-neuraminidase, β-glucuronidase) were measured in milk samples from six cows with different SCC at quarter level. Glycosidase activities were related to levels of free sugars as assessed by NMR spectroscopy. NAGase, β-glucuronidase, β-glucosidase, α-galactosidase and β-galactosidase were the enzymes to increase most with SCC. Furthermore, free fucose level was positively correlated with SCC (r = 0.72, P < 0.0001). The same tendency was observed for sialic acid, whereas galactose and N-acetyl-hexosamine were found to increase consistently in quarters of mastitic cows regardless of SCC. This indicates different potential origins of responsible enzymes and further illustrates the potential effects of milk glycosidases on the inherent glycans present in milk.

Acceptive Immunity: The Role of Fucosylated Glycans in Human Host-Microbiome Interactions.

The growth in the number of chronic non-communicable diseases in the second half of the past century and in the first two decades of the new century is largely due to the disruption of the relationship between the human body and its symbiotic microbiota, and not pathogens. The interaction of the human immune system with symbionts is not accompanied by inflammation, but is a physiological norm. This is achieved via microbiota control by the immune system through a complex balance of pro-inflammatory and suppressive responses, and only a disturbance of this balance can trigger pathophysiological mechanisms. This review discusses the establishment of homeostatic relationships during immune system development and intestinal bacterial colonization through the interaction of milk glycans, mucins, and secretory immunoglobulins. In particular, the role of fucose and fucosylated glycans in the mechanism of interactions between host epithelial and immune cells is discussed.

Structure and evolution of the bifidobacterial carbohydrate metabolism proteins and enzymes.

Bifidobacteria have attracted significant attention because they provide health-promoting effects in the human gut. In this review, we present a current overview of the three-dimensional structures of bifidobacterial proteins involved in carbohydrate uptake, degradation, and metabolism. As predominant early colonizers of the infant's gut, distinct bifidobacterial species are equipped with a panel of transporters and enzymes specific for human milk oligosaccharides (HMOs). Interestingly, Bifidobacterium bifidum and Bifidobacterium longum possess lacto-N-biosidases with unrelated structural folds to release the disaccharide lacto-N-biose from HMOs, suggesting the convergent evolution of this activity from different ancestral proteins. The crystal structures of enzymes that confer the degradation of glycans from the mucin glycoprotein layer provide a structural basis for the utilization of this sustainable nutrient in the gastrointestinal tract. The utilization of several plant dietary oligosaccharides has been studied in detail, and the prime importance of oligosaccharide-specific ATP-binding cassette (ABC) transporters in glycan utilisations by bifidobacteria has been revealed. The structural elements underpinning the high selectivity and roles of ABC transporter binding proteins in establishing competitive growth on preferred oligosaccharides are discussed. Distinct ABC transporters are conserved across several bifidobacterial species, e.g. those targeting arabinoxylooligosaccharide and α-1,6-galactosides/glucosides. Less prevalent transporters, e.g. targeting β-mannooligosaccharides, may contribute to the metabolic specialisation within Bifidobacterium. Some bifidobacterial species have established symbiotic relationships with humans. Structural studies of carbohydrate-utilizing systems in Bifidobacterium have revealed the interesting history of molecular coevolution with the host, as highlighted by the early selection of bifidobacteria by mucin and breast milk glycans.

Effective Separation of Human Milk Glycosides using Carbon Dioxide Supercritical Fluid Chromatography.

Carbohydrate purification remains problematic due to the intrinsic diversity of structural isomers present in nature. Although liquid chromatography-based techniques are suitable for analyzing or preparing most glycan structures acquired either from natural sources or through chemical or enzymatic synthesis, the separation of regioisomers or linkage isomers with a clear resolution remains challenging. Herein, a carbon dioxide supercritical fluid chromatography (SFC) method was devised to resolve 18 human milk glycosides: oligomers (disaccharides to hexasaccharides), fucosylated regioisomers (lacto-N-fucopentaose I, III, and V; lacto-N-neofucopentaose V; lacto-N-difucohexaose III; blood group H1 antigen; and TF-LNnT), and connectivity isomers (lacto-N-tetraose/lacto-N-neotetraose and para-lacto-N-hexaose/para-lacto-N-neohexaose/type-1 hexasaccharide). The analysis of these glycosides represents a major limitation associated with conventional carbohydrate analysis. The unprecedented resolution achieved by the SFC method indicates the suitability of this key technology for revealing complex human milk glycomes.

Infant gut microbiota modulation by human milk disaccharides in humanized microbiome mice

ABSTRACT Human milk glycans present a unique diversity of structures that suggest different mechanisms by which they may affect the infant microbiome development. A humanized mouse model generated by infant fecal transplantation was utilized here to evaluate the impact of fucosyl-α1,3-GlcNAc (3FN), fucosyl-α1,6-GlcNAc, lacto-N-biose (LNB) and galacto-N-biose on the fecal microbiota and host–microbiota interactions. 16S rRNA amplicon sequencing showed that certain bacterial genera significantly increased (Ruminococcus and Oscillospira) or decreased (Eubacterium and Clostridium) in all disaccharide-supplemented groups. Interestingly, cluster analysis differentiates the consumption of fucosyl-oligosaccharides from galactosyl-oligosaccharides, highlighting the disappearance of Akkermansia genus in both fucosyl-oligosaccharides. An increment of the relative abundance of Coprococcus genus was only observed with 3FN. As well, LNB significantly increased the relative abundance of Bifidobacterium, whereas the absolute levels of this genus, as measured by quantitative real-time PCR, did not significantly increase. OTUs corresponding to the species Bifidobacterium longum, Bifidobacterium adolescentis and Ruminococcus gnavus were not present in the control after the 3-week intervention, but were shared among the donor and specific disaccharide groups, indicating that their survival is dependent on disaccharide supplementation. The 3FN-feeding group showed increased levels of butyrate and acetate in the colon, and decreased levels of serum HDL-cholesterol. 3FN also down-regulated the pro-inflammatory cytokine TNF-α and up-regulated the anti-inflammatory cytokines IL-10 and IL-13, and the Toll-like receptor 2 in the large intestine tissue. The present study revealed that the four disaccharides show efficacy in producing beneficial compositional shifts of the gut microbiota and in addition, the 3FN demonstrated physiological and immunomodulatory roles.

Associations between human milk oligosaccharides (HMOs) and eating behaviour in Hispanic infants at 1 and 6 months of age

Human milk oligosaccharides (HMOs) are naturally occurring glycans in human breast milk that act as prebiotics in the infant gut. Prebiotics have been demonstrated to suppress appetite in both adults and children. Therefore, HMOs may affect infant eating behaviour. To determine if HMOs in breast milk are associated with eating behaviour in Hispanic infants. Cross-sectional analysis of a prospective cohort of Hispanic mother-infant dyads (1-month, n = 157; 6-months, n = 69). Breast milk samples were screened for 19 HMOs using high pressure liquid chromatography, and eating behaviour was assessed using the Baby Eating Behaviour Questionnaire (BEBQ). We conducted multiple linear regressions to examine associations between HMOs and BEBQ scores, adjusted for maternal pre-pregnancy BMI, infant sex, birthweight, delivery mode and number of breastfeedings per day. We stratified by HMO secretor status-a genetic determinant of the types of HMOs produced. At 1 month, LNnT (lacto-N-neotetraose; P = .04) was negatively associated with food responsiveness in the total sample, while DFLNT (difucosyllacto-N-tetrose; P = .03) and DSLNT (disialyl-LNT; P = .04) were negatively associated with food responsiveness in secretors only. At 6 months, LSTc (sialyllacto-N-tetraose c; P = .01), FLNH (fucosyllacto-N-hexaose; P = .03), LNH (lacto-N-hexaose; P = .006) and DSLNH (disialyllacto-N-hexaose; P = .05) were positively associated with food responsiveness in both the total sample and secretors only. We found several HMOs that were both positively and negatively associated with infant food responsiveness, which is a measure of drive to eat.

High levels of fucosylation and sialylation of milk N‐glycans from mothers with gestational diabetes mellitus alter the offspring gut microbiome and immune balance in mice

Gestational diabetes mellitus (GDM) is significantly associated with allergen sensitization in early childhood, and this may influence the gut microbiome and immune system of the children. In addition to mother-to-child transmission of microbes, milk glycans play a pivotal role in shaping the gut microbiome of infants. A previous study has demonstrated alterations in the major milk N-glycans of mothers with GDM. However, the impact of these changes on the gut microbiome and immune response of the neonates has yet to be studied. Here, we aimed to compare the glycosylation levels of various milk glycans between normal and GDM mice, and to characterize the intestinal microbiome and immune responses of the offspring after weaning. We found that GDM mouse milk contained significantly higher concentrations of fucosylated and sialylated N-glycans than control mice, but there was no difference in the concentration of milk oligosaccharides between the groups. The differences in milk N-glycans had direct effects on the intestinal microbiome of the offspring, which in turn affected their immune response upon challenge with ovalbumin (OVA), with disruptions in the Th1/Th2 and Th17/Treg cell balances. This study lays the foundation for further research and development of specific nutritional care for the offspring of GDM mothers.

Structural basis for broad substrate specificity of UDP-glucose 4-epimerase in the human milk oligosaccharide catabolic pathway of Bifidobacterium longum

Infant gut-associated bifidobacteria has a metabolic pathway that specifically utilizes lacto-N-biose I (Gal-β1,3-GlcNAc) and galacto-N-biose (Gal-β1,3-GalNAc) from human milk and mucin glycans. UDP-glucose 4-epimerase (GalE) from Bifidobacterium longum (bGalE) catalyzes epimerization reactions of UDP-Gal into UDP-Glc and UDP-GalNAc into UDP-GlcNAc with the same level of activity that is required to send galacto-hexoses into glycolysis. Here, we determined the crystal structures of bGalE in three ternary complex forms: NAD+/UDP, NAD+/UDP-GlcNAc, and NAD+/UDP-Glc. The broad specificity of bGalE was explained by structural features of the binding pocket for the N-acetyl or C2 hydroxy group of the substrate. Asn200 is located in a pocket of the C2 group, and its side chain adopts different conformations in the complex structures with UDP-Glc and UDP-GlcNAc. On the other side, Cys299 forms a large pocket for the C5 sugar ring atom. The flexible C2 pocket and the large C5 pocket of bGalE are suitable for accommodating both the hydroxy and N-acetyl groups of the substrate during sugar ring rotation in the catalytic cycle. The substrate specificity and active site structure of bGalE were distinct from those of Esherichia coli GalE but similar to those of human GalE.

Fucosylated Human Milk Oligosaccharides and N-Glycans in the Milk of Chinese Mothers Regulate the Gut Microbiome of Their Breast-Fed Infants during Different Lactation Stages.

The milk glycobiome has a significant impact on the gut microbiota of infants, which plays a pivotal role in health and development. Fucosylated human milk oligosaccharides (HMOs) and N-glycans on milk proteins are beneficial for the development of healthy gut microbiota, and the fucosylation levels of these glycans can be affected by the maternal fucosyltransferase 2 gene (FUT2). Here, we present results of longitudinal research on paired milk and stool samples from 56 Chinese mothers (CMs) and their breast-fed children. Changes of HMOs and fucosylated N-glycans in milk of CMs at different lactation stages were detected, which allowed characterization of the major differences in milk glycans and consequential effects on the gut microbiome of infants according to maternal FUT2 status. Significant differences in the abundance of total and fucosylated HMOs between secretor and nonsecretor CMs were noted, especially during early lactation. Despite a tendency toward decreasing milk protein concentrations, the fucosylation levels of milk N-glycans increased during late lactation. The changes in the levels of fucosylated HMOs and milk N-glycans were highly correlated with the growth of Bifidobacterium spp. and Lactobacillus spp. in the gut of infants during early and later lactation, respectively. Enriched expression of genes encoding glycoside hydrolases, glycosyl transferases, ATP-binding cassette (ABC) transporters, and permeases in infants fed by secretor CMs contributed to the promotion of these bacteria in infants. Our data highlight the important role of fucosylated milk glycans in shaping the gut microbiome of infants and provide a solid foundation for development of "personalized" nutrition for Chinese infants. IMPORTANCE Human milk glycans provide a broad range of carbon sources for gut microbes in infants. Levels of protein glycosylation in human milk vary during lactation and may also be affected by the stages of gestation and lactation and by the secretor status of the mother. This was the first study to evaluate systematically dynamic changes in human milk oligosaccharides and fucosylated N-glycans in the milk of Chinese mothers with different secretor statuses during 6 months of lactation. Given the unique single nucleotide polymorphism site (rs1047781, A385T) on the fucosyltransferase 2 gene among Chinese populations, our report provides a specific insight into the milk glycobiome of Chinese mothers, which may exert effects on the gut microbiota of infants that differ from findings from other study cohorts.