Bifidobacterium longum subsp. infantis ATCC 15697 utilizes several small-mass neutral human milk oligosaccharides (HMOs), several of which are fucosylated. Whereas previous studies focused on endpoint consumption, a temporal glycan consumption profile revealed a time-dependent effect. Specifically, among preferred HMOs, tetraose was favored early in fermentation, with other oligosaccharides consumed slightly later. In order to utilize fucosylated oligosaccharides, ATCC 15697 possesses several fucosidases, implicating GH29 and GH95 ␣-L-fucosidases in a gene cluster dedicated to HMO metabolism. Evaluation of the biochemical kinetics demonstrated that ATCC 15697 expresses three fucosidases with a high turnover rate. Moreover, several ATCC 15697 fucosidases are active on the linkages inherent to the HMO molecule. Finally, the HMO cluster GH29 ␣-L-fucosidase possesses a crystal structure that is similar to previously characterized fucosidases.T he genus Bifidobacterium is frequently overrepresented in the breast-fed infant colon relative to its appearance in adults, where these organisms are believed to benefit their host through nutrient supplementation, participating in host energy cycling and binding to preferred host receptor molecules otherwise available to pathogens (12). Selective growth of bifidobacteria has been attributed to utilization of oligosaccharides abundant in human milk (10 to 20 g/liter) that present complex structures resistant to infant digestion (17, 35). Approximately 200 species of human milk oligosaccharides (HMOs) have been characterized that are composed of glucose, galactose, N-acetylglucosamine, and often fucose and/or sialic acid residues via several glycosidic linkages (25). The HMO core is typically elongated from a lactosyl reducing end (Gal␤1-4Glc) that is linked via ␤1-3 (or ␤1-6 in branched molecules) to serial lacto-N-biose I units (Gal␤1-3GlcNAc) or lactosamine (Gal␤1-4GlcNAc) with a degree of polymerization of Ն4.As with other fucosylated glycoconjugates, ␣1-2/3/4 fucosyl moieties often shield HMOs from digestion unless this linkage at the nonreducing terminus is first cleaved. Similarly, acidic HMOs or milk sialyloligosaccharides (MSOs) obstruct enzymatic degradation with sialyl residues via ␣2-3/6 linkages. Removal of these termini is postulated to initiate bacterial catabolism of HMOs (2,8,30). To this end, it has been recently demonstrated that Bifidobacterium longum subsp. infantis ATCC 15697 utilizes milk sialyloligosaccharides via a sialidase encoded within a large gene cluster dedicated to HMO metabolism (30).Previous research conducted on bifidobacterial metabolism of fucosylated oligosaccharides identified a Bifidobacterium bifidum ␣1-2-L-fucosidase that exhibited an atypical inverting mechanism (glycoside hydrolase [GH] family 95), termed AfcA (10,22). Inverting glycoside hydrolases modify anomeric stereochemistry via a single nucleophilic displacement, mechanistically contrasting with retaining enzymes, which maintain the anomeric configuration through catalysis of ...

The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip. In all, 38 N-glycan compositions were observed in the human milk sample while the bovine milk sample revealed 51 N-glycan compositions. These numbers translate to over a hundred compounds when isomers are considered and point to the complexity of the mixture. High mannose, neutral, and sialylated complex/hybrid glycans were observed in both milk sources. Although NeuAc sialylation was observed in both milk samples, the NeuGc residue was only observed in bovine milk and marks a major difference between human and bovine milks. To the best of our knowledge, this study is the first MS based confirmation of NeuGc in milk protein bound glycans as well as the first comprehensive N-glycan profile of bovine milk proteins. Tandem MS was necessary for resolving complications presented by the fact that (NeuGc:Fuc) corresponds to the exact mass of (NeuAc:Hex). Comparison of the relative distribution of the different glycan types in both milk sources was possible via their abundances. While the human milk analysis revealed a 6% high mannose, 57% sialylation, and 75% fucosylation distribution, a 10% high mannose, 68% sialylation, and 31% fucosylation distribution was observed in the bovine milk analysis. Comparison with the free milk oligosaccharides yielded low sialylation and high fucosylation in human, while high sialylation and low fucosylation are found in bovine. The results suggest that high fucosylation is a general trait in human, while high sialylation and low fucosylation are general features of glycosylation in bovine milk.

Lactating mothers secrete milk sialyloligosaccharides (MSOs) that function as anti-adhesives once provided to the neonate. Particular infant-associated commensals, such as Bifidobacterium longum subsp. infantis, consume neutral milk oligosaccharides, although their ability to utilize acidic oligosaccharides has not been assessed. Temporal glycoprofiling of acidic HMO consumed during fermentation demonstrated a single composition, with several isomers, corresponding to sialylated lacto-Ntetraose. To utilize MSO, B. longum subsp. infantis deploys a sialidase that cleaves ␣2-6 and ␣2-3 linkages. NanH2, encoded within the HMO catabolic cluster is up-regulated during HMO fermentation and is active on sialylated lacto-N-tetraose. These results demonstrate that commensal microorganisms do utilize MSO, a substrate that may be enriched in the distal gastrointestinal tract.Soluble oligosaccharides often exceed protein concentrations in human milk, although their biological role remains poorly defined. These heterogeneous carbohydrates consist of glucose, galactose, N-acetylglucosamine, and frequently fucose and/or N-acetyl-D-neuraminic acid (Neu5Ac or sialic acid) residues via several potential glycosidic linkages. The human milk oligosaccharide (HMO) 4 core is typically elongated from a lactose reducing end (Gal␤1-4Glc) with iterative Gal␤1-3/ 4GlcNAc units to compose linear or branched oligosaccharides with a degree of polymerization Ն 4. ␣1-2/3/4 Fucosylation adds structural complexity and may shield the HMO backbone from exo-glycosidase digestion. Similarly, enzymatic degradation of acidic HMOs or milk sialyloligosaccharides (MSOs) requires cleavage of terminal ␣2-6-and ␣2-3-linked sialyl moieties (1). Human milk is a rich source of sialylated glycans, previously determined to be over 40 structures (of over 200 total HMOs) representing nearly 16% of soluble oligosaccharide abundances (2, 3).In contrast to lactose, the structural organization of HMOs resist host digestive enzymes, thus are introduced intact to microbial communities established along the nursing infant gastrointestinal tract (GIT) (4). Accordingly, HMOs are potent molecular arbiters at the mammalian-microbial interface as they modulate epithelial surface glycan expression and may modulate systemic immune responses (5). Moreover, HMOs limit pathogen colonization by mimicking vulnerable host epitopes thus competing for microbial adhesins. A well-studied example is the inhibition of campylobacter-induced diarrhea in infants by ␣1-2 fucosylated HMO (6, 7). Likewise, sialylated glycans are bound by Helicobacter pylori and are known to mitigate Streptococcus pneumoniae adherence to the nasopharyngeal mucosa (8 -10). In addition, sialylated milk glycoproteins confer further protection by neutralizing infectious particles such as rotavirus (11).With a few exceptions, the prominence of sialic acids to eukaryotic biology emerged with the deuterostomes and thus microbial interactions denote an evolved commensal, pathogenic, or saprotrophic relationship with anim...

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Gangliosides are anionic glycosphingolipids widely distributed in vertebrate tissues and fluids. Their structural and quantitative expression patterns depend on phylogeny and are distinct down to the species level. In milk, gangliosides are exclusively associated with the milk fat globule membrane. They may participate in diverse biological processes but more specifically to host-pathogen interactions. However, due to the molecular complexities, the analysis needs extensive sample preparation, chromatographic separation, and even chemical reaction, which makes the process very complex and time-consuming. Here, we describe a rapid profiling method for bovine and human milk gangliosides employing matrix-assisted desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS). Prior to the analyses of biological samples, milk ganglioside standards GM3 and GD3 fractions were first analyzed in order to validate this method. High mass accuracy and high resolution obtained from MALDI FTICR MS allow for the confident assignment of chain length and degree of unsaturation of the ceramide. For the structural elucidation, tandem mass spectrometry (MS/MS), specifically as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) were employed. Complex ganglioside mixtures from bovine and human milk were further analyzed with this method. The samples were prepared by two consecutive chloroform/methanol extraction and solid phase extraction. We observed a number of differences between bovine milk and human milk. The common gangliosides in bovine and human milk are NeuAc-NeuAc-Hex-Hex-Cer (GD3) and NeuAc-Hex-Hex-Cer (GM3); whereas, the ion intensities of ganglioside species are different between two milk samples. Kendrick mass defect plot yields grouping of ganglioside peaks according to their structural similarities. Gangliosides were further probed by tandem MS to confirm the compositional and structural assignments. We found that only in human milk gangliosides was the ceramide carbon always even numbered, which is consistent with the notion that differences in the oligosaccharide and the ceramide moieties confer to their physiological distinctions.