Protein glycosylation and other post-translational modifications are involved in potentially all aspects of human growth and development. Defective glycosylation has adverse effects on human ...physiological conditions and accompanies many chronic and infectious diseases. Altered glycosylation can occur at the onset and/or during tumor progression. Identifying these changes at early disease stages may aid in making decisions regarding treatments, as early intervention can greatly enhance survival. This review highlights some of the efforts being made to identify N- and O-glycosylation profile shifts in cancer using mass spectrometry. The analysis of single or panels of potential glycoprotein cancer markers are covered. Other emerging technologies such as global glycan release and site-specific glycosylation analysis and quantitation are also discussed.
Graphical Abstract
Steps involved in the biomarker discovery
The mammalian intestine is host to a microbial community that prevents pathogen expansion through unknown mechanisms, while antibiotic treatment can increase susceptibility to enteric pathogens. Here ...we show that streptomycin treatment depleted commensal, butyrate-producing Clostridia from the mouse intestinal lumen, leading to decreased butyrate levels, increased epithelial oxygenation, and aerobic expansion of Salmonella enterica serovar Typhimurium. Epithelial hypoxia and Salmonella restriction could be restored by tributyrin treatment. Clostridia depletion and aerobic Salmonella expansion were also observed in the absence of streptomycin treatment in genetically resistant mice but proceeded with slower kinetics and required the presence of functional Salmonella type III secretion systems. The Salmonella cytochrome bd-II oxidase synergized with nitrate reductases to drive luminal expansion, and both were required for fecal-oral transmission. We conclude that Salmonella virulence factors and antibiotic treatment promote pathogen expansion through the same mechanism: depletion of butyrate-producing Clostridia to elevate epithelial oxygenation, allowing aerobic Salmonella growth.
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•Salmonella-induced colitis drives a depletion of butyrate-producing Clostridia•Antibiotic-mediated depletion of Clostridia increases colonocyte oxygenation•Increased epithelial oxygenation drives an aerobic post-antibiotic pathogen expansion•A respiration-driven Salmonella expansion in the gut is required for transmission
Paradoxically, antibiotic treatment can promote relapse of Salmonella gastroenteritis. Rivera-Chávez et al. show that antibiotic treatment lowers colonization resistance by depleting butyrate-producing Clostridia. Decreased butyrate availability increases epithelial oxygenation, thereby fueling aerobic pathogen expansion in the gut lumen. Aerobic respiration synergizes with nitrate respiration to drive fecal-oral transmission of Salmonella.
Glycomic and glycoproteomic analyses involve the characterization of oligosaccharides (glycans) conjugated to proteins. Glycans are produced through a complicated nontemplate driven process involving ...the competition of enzymes that extend the nascent chain. The large diversity of structures, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies of glycans all conspire to make the analysis arguably much more difficult than any other biopolymer. Furthermore, the large number of glycoforms associated with a specific protein site makes it more difficult to characterize than any post-translational modification. Nonetheless, there have been significant progress, and advanced separation and mass spectrometry methods have been at its center and the main reason for the progress. While glycomic and glycoproteomic analyses are still typically available only through highly specialized laboratories, new software and workflow is making it more accessible. This review focuses on the role of mass spectrometry and separation methods in advancing glycomic and glycoproteomic analyses. It describes the current state of the field and progress toward making it more available to the larger scientific community.
Oligosaccharides are abundant in human milk. Production of these highly diverse structures requires significant energy expenditure by the mother and yet these human milk oligosaccharides offer no ...direct nutritive value to her infant. A primary function of human milk oligosaccharides is to shape the infant's intestinal microbiota with life-long consequences. Bifidobacterium longum subspecies infantis (B. infantis) is unique among gut bacteria in its prodigious capacity to digest and consume any human milk oligosaccharide structure, the result of a large repertoire of bacterial genes encoding an array of glycosidases and oligosaccharide transporters not found in other bacterial species. In vitro, B. infantis grows better than other bacterial strains in the presence of human milk oligosaccharides, displays anti-inflammatory activity in premature intestinal cells, and decreases intestinal permeability. In premature infants, B. infantis given in combination with human milk increases B. infantis and decreases Enterobacteriaceae in the feces. Probiotics containing B. infantis decrease the risk of necrotizing enterocolitis in premature infants. Colonization with B. infantis is also associated with increased vaccine responses. Probiotic organisms have historically been selected based on ease of production and stability. The advantages of B. infantis, selected through coevolution with human milk glycans, present an opportunity for focused manipulation of the infant intestinal microbiota.
Human milk contains an unexpected abundance and diversity of complex oligosaccharides apparently indigestible by the developing infant and instead targeted to its cognate gastrointestinal microbiota. ...Recent advances in mass spectrometry-based tools have provided a view of the oligosaccharide structures produced in milk across stages of lactation and among human mothers. One postulated function for these oligosaccharides is to enrich a specific "healthy" microbiota containing bifidobacteria, a genus commonly observed in the feces of breast-fed infants. Isolated culture studies indeed show selective growth of infant-borne bifidobacteria on milk oligosaccharides or core components therein. Parallel glycoprofiling documented that numerous Bifidobacterium longum subsp. infantis strains preferentially consume small mass oligosaccharides that are abundant early in the lactation cycle. Genome sequencing of numerous B. longum subsp. infantis strains shows a bias toward genes required to use mammalian-derived carbohydrates by comparison with adult-borne bifidobacteria. This intriguing strategy of mammalian lactation to selectively nourish genetically compatible bacteria in infants with a complex array of free oligosaccharides serves as a model of how to influence the human supraorganismal system, which includes the gastrointestinal microbiota.
Newborns are colonized with an intestinal microbiota shortly after birth, but the factors governing the retention and abundance of specific microbial lineages are unknown. Nursing infants consume ...human milk oligosaccharides (HMOs) that pass undigested to the distal gut, where they may be digested by microbes. We determined that the prominent neonate gut residents,
Bacteroides thetaiotaomicron and
Bacteroides fragilis, induce the same genes during HMO consumption that are used to harvest host mucus glycans, which are structurally similar to HMOs. Lacto-
N-neotetraose, a specific HMO component, selects for HMO-adapted species such as
Bifidobacterium infantis, which cannot use mucus, and provides a selective advantage to
B. infantis in vivo when biassociated with
B. thetaiotaomicron in the gnotobiotic mouse gut. This indicates that the complex oligosaccharide mixture within HMOs attracts both mutualistic mucus-adapted species and HMO-adapted bifidobacteria to the infant intestine that likely facilitate both milk and future solid food digestion.
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Bacteroides are adept at consuming human milk oligosaccharides ►
Bacteroides induce mucus-utilization genes to consume human milk oligosaccharides ► Lacto-
N-neotetraose selects for
Bifidobacteria relative to
Bacteroides in vivo
Glycosylation is an important post-translational modification of proteins present in the vast majority of human proteins. For this reason, they are potentially new sources of biomarkers and active ...targets of therapeutics and vaccines. However, the absence of a biosynthetic template as in the genome and the general complexity of the structures have limited the development of methods for comprehensive structural analysis. Even now, the exact structures of many abundant N-glycans in serum are not known. Structural elucidation of oligosaccharides remains difficult and time-consuming. Here, we introduce a means of rapidly identifying released N-glycan structures using their accurate masses and retention times based on a glycan library. This serum glycan library, significantly expanded from a previous one covering glycans released from a handful of serum glycoproteins, has more than 170 complete and partial structures and constructed instead from whole serum. The method employs primarily nanoflow liquid chromatography and accurate mass spectrometry. The method allows us to readily profile N-glycans in biological fluids with deep structural analysis. This approach is used to determine the relative abundances and variations in the N-glycans from several individuals providing detailed variations in the abundances of the important N-glycans in blood.
Abstract Herein we will review the role of glycans in the immune system. Specific topics covered include: the glycosylation sites of IgE, IgM, IgD, IgE, IgA, and IgG; how glycans can encode “self” ...identity by functioning as either danger associated molecular patterns (DAMPs) or self-associated molecular patterns (SAMPs); the role of glycans as markers of protein integrity and age; how the glycocalyx can dictate the migration pattern of immune cells; and how the combination of Fc N-glycans and Ig isotype dictate the effector function of immunoglobulins. We speculate that the latter may be responsible for the well-documented association between alterations of the serum glycome and autoimmunity. Due to technological limitations, the extent of these autoimmune-associated glycan alterations and their role in disease pathophysiology has not been fully elucidated. Thus, we also review the current technologies available for glycan analysis, placing an emphasis on Multiple Reaction Monitoring (MRM), a rapid high-throughput technology that has great potential for glycan biomarker research. Finally, we put forth The Altered Glycan Theory of Autoimmunity , which states that each autoimmune disease will have a unique glycan signature characterized by the site-specific relative abundances of individual glycan structures on immune cells and extracellular proteins, especially the site-specific glycosylation patterns of the different immunoglobulin(Ig) classes and subclasses.
Perturbation of the gut-associated microbial community may underlie many human illnesses, but the mechanisms that maintain homeostasis are poorly understood. We found that the depletion of ...butyrate-producing microbes by antibiotic treatment reduced epithelial signaling through the intracellular butyrate sensor peroxisome proliferator–activated receptor γ (PPAR-γ). Nitrate levels increased in the colonic lumen because epithelial expression of Nos2, the gene encoding inducible nitric oxide synthase, was elevated in the absence of PPAR-γ signaling. Microbiota-induced PPAR-γ signaling also limits the luminal bioavailability of oxygen by driving the energy metabolism of colonic epithelial cells (colonocytes) toward β-oxidation. Therefore, microbiota-activated PPAR-γ signaling is a homeostatic pathway that prevents a dysbiotic expansion of potentially pathogenic Escherichia and Salmonella by reducing the bioavailability of respiratory electron acceptors to Enterobacteriaceae in the lumen of the colon.
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