Analysis and role of oligosaccharides in milk

Milk is an important fluid in glycobiology because it contains a number of short carbohydrate chains either free or as glycoconjugates. These compounds as a class are the most abundant component and benefit the infant by developing and maintaining the infant's gut flora. New and emerging methods for oligosaccharide analysis have been developed to study milk. These methods allow for the rapid profiling of oligosaccharide mixtures with quantitation. With these tools, the role of oligosaccharide in milk is being understood. They further point to how oligosaccharide analysis can be performed, which until now has been very difficult and have lagged significantly those of other biopolymers. [BMB Reports 2012; 45(8): 442-451].     

Profiling with structural elucidation of the neutral and anionic O-linked oligosaccharides in the egg jelly coat of Xenopus laevis by Fourier transform mass spectrometry

A strategic method with high speed and sensitivity is outlined for the analysis of mucin-type oligosaccharide from the jelly coat of Xenopus laevis. The method relies primarily on mass spectrometric techniques, in this case matrix-assisted laser desorption/ionization Fourier-transform mass spectrometry (MALDI-FTMS) and collision-induced dissociation (CID). Separation with isolation of the oligosaccharides was streamlined to couple well with mass spectrometry allowing the rapid determination of all detectable components from both neutral and anionic species. Partial structures of anionic components, composed primarily of sulfate esters, were obtained with CID. For neutral species, a method that allowed the complete structural determination using mass spectrometry was used. The method builds on the structure of small number of known compounds to determine unknown structures from the same biological source. In this example, a small number of oligosaccharides, elucidated previously by NMR, were used to develop a set of substructural motifs that were characterized by CID. The presence of the motifs in the CID spectra were then used to determine the structures of unknown compounds that were in abundances too small for NMR analysis.     

Proteomic Analysis of Bifidobacterium longum subsp. infantis Reveals the Metabolic Insight on Consumption of Prebiotics and Host Glycans

Bifidobacterium longum subsp. infantis is a common member of the intestinal microbiota in breast-fed infants and capable of metabolizing human milk oligosaccharides (HMO). To investigate the bacterial response to different prebiotics, we analyzed both cell wall associated and whole cell proteins in B. infantis. Proteins were identified by LC-MS/MS followed by comparative proteomics to deduce the protein localization within the cell. Enzymes involved in the metabolism of lactose, glucose, galactooligosaccharides, fructooligosaccharides and HMO were constitutively expressed exhibiting less than two-fold change regardless of the sugar used. In contrast, enzymes in N-Acetylglucosamine and sucrose catabolism were induced by HMO and fructans, respectively. Galactose-metabolizing enzymes phosphoglucomutase, UDP-glucose 4-epimerase and UTP glucose-1-P uridylytransferase were expressed constitutively, while galactokinase and galactose-1-phosphate uridylyltransferase, increased their expression three fold when HMO and lactose were used as substrates for cell growth. Cell wall-associated proteomics also revealed ATP-dependent sugar transport systems associated with consumption of different prebiotics. In addition, the expression of 16 glycosyl hydrolases revealed the complete metabolic route for each substrate. Mucin, which possesses O-glycans that are structurally similar to HMO did not induced the expression of transport proteins, hydrolysis or sugar metabolic pathway indicating B. infantis do not utilize these glycoconjugates.     

Genetic Ablation of Butyrate Utilization Attenuates Gastrointestinal Salmonella Disease

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In Vitro Fermentation of Breast Milk Oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri

It has been proposed that human milk oligosaccharides (HMO) function as a prebiotic for bifidobacteria, yet this activity has not been adequately investigated. In this study, Bifidobacterium infantis was shown to ferment purified HMO as a sole carbon source, while another gut commensal, Lactobacillus gasseri, did not ferment HMO. Our results support the hypothesis that HMO selectively amplify bacterial populations in the infant intestine.     

The glycolyzer: Automated glycan annotation software for high performance mass spectrometry and its application to ovarian cancer glycan biomarker discovery

Human serum glycomics is a promising method for finding cancer biomarkers but often lacks the tools for streamlined data analysis. The Glycolyzer software incorporates a suite of analytic tools capable of identifying informative glycan peaks out of raw mass spectrometry data. As a demonstration of its utility, the program was used to identify putative biomarkers for epithelial ovarian cancer from a human serum sample set. A randomized, blocked, and blinded experimental design was used on a discovery set consisting of 46 cases and 48 controls. Retrosynthetic glycan libraries were used for data analysis and several significant candidate glycan biomarkers were discovered via hypothesis testing. The significant glycans were attributed to a glycan family based on glycan composition relationships and incorporated into a linear classifier motif test. The motif test was then applied to the discovery set to evaluate the disease state discrimination performance. The test provided strongly predictive results based on receiver operator characteristic curve analysis. The area under the receiver operator characteristic curve was 0.93. Using the Glycolyzer software, we were able to identify a set of glycan biomarkers that highly discriminate between cases and controls, and are ready to be formally validated in subsequent studies.     

Glycosylation of human milk lactoferrin exhibits dynamic changes during early lactation enhancing its role in pathogenic bacteria-host interactions

Human milk lactoferrin (hmLF) is the most abundant glycoprotein present in human milk and displays a broad range of protective functions in the gut of newborn infants. hmLF is N-glycosylated, but little is known about the lactation stage-related development of the glycosylation phenotype. hmLF glycosylation from milk samples from five donors during the first 10 weeks of lactation was assessed and observed to be more diverse than previously reported. During this period dynamic changes in glycosylation were observed corresponding to a decrease in glycosylation in the second week followed by an increase in total glycosylation as well as higher order fucosylation thereafter. Gene expression analysis was performed in milk somatic cells from a sixth subject. It was found that fucosyltransferase expression increased during entire period, whereas expression of genes for the oligosaccharyl transferase complex decreased in the second week. The effect of hmLF glycosylation was examined for the protein's ability to affect bacterial binding to epithelial cells. hmLF significantly inhibited pathogen adhesion and purified hmLF glycans significantly reduced Salmonella invasion of colonic epithelial cells to levels associated with non-invasive deletion mutants. This study indicates that hmLF glycosylation is tightly regulated by gene expression and that glyco-variation is involved in modulating pathogen association.     

Methods for the quantitation of human milk oligosaccharides in bacterial fermentation by mass spectrometry

Oligosaccharides are the third most abundant component in human milk. In the past decades, it became apparent that they would be able to protect against pathogens and participate in the development of the gut microflora for infants. However, their role in infants' nutrition and development remains poorly understood. To better understand this function, it is extremely important to have a quantitative tool for profiling oligosaccharides. In this article, we show the development of a method to quantitatively differentiate the relative amounts of oligosaccharides fermented by different intestinal bacteria. To determine the oligosaccharide consumption, bacteria were grown in a medium using human milk oligosaccharides (HMOs) as the only carbon source purified from breast milk and further analyzed by matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS). A method using an internal deuterium-labeled standard was developed and compared with an external standard method, with the internal standard method giving better precision and unambiguous measurements than the external standard method and providing to be a novel and robust tool for following bacterial fermentation of milk oligosaccharides.