Search

Bifidobacteria are among the first colonizers of the infant gut, and human milk oligosaccharides (HMOs) in breastmilk are instrumental for the formation of a bifidobacteria-rich microbiota. However, little is known about the assembly of bifidobacterial communities. Here, by applying assembly theory to a community of four representative infant-gut associated Bifidobacterium species that employ varied strategies for HMO consumption, we show that arrival order and sugar consumption phenotypes significantly affected community formation. Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, two avid HMO consumers, dominate through inhibitory priority effects. On the other hand, Bifidobacterium breve, a species with limited HMO-utilization ability, can benefit from facilitative priority effects and dominates by utilizing fucose, an HMO degradant not utilized by the other bifidobacterial species. Analysis of publicly available breastfed infant faecal metagenome data showed that the observed trends for B. breve were consistent with our in vitro data, suggesting that priority effects may have contributed to its dominance. Our study highlights the importance and history dependency of initial community assembly and its implications for the maturation trajectory of the infant gut microbiota., 母乳栄養児の腸内におけるビフィズス菌コミュニティー形成には先住効果が大きな影響を及ぼす --ヒトミルクオリゴ糖利用能力の低いビフィズス菌B. breveが優勢となる仕組み--. 京都大学プレスリリース. 2022-07-27.

Mucinolytic bacteria modulate host–microbiota symbiosis and dysbiosis through their ability to degrade mucin O-glycans. However, how and to what extent bacterial enzymes are involved in the breakdown process remains poorly understood. Here we focus on a glycoside hydrolase family 20 sulfoglycosidase (BbhII) from Bifidobacterium bifidum, which releases N-acetylglucosamine-6-sulfate from sulfated mucins. Glycomic analysis showed that, in addition to sulfatases, sulfoglycosidases are involved in mucin O-glycan breakdown in vivo and that the released N-acetylglucosamine-6-sulfate potentially affects gut microbial metabolism, both of which were also supported by a metagenomic data mining analysis. Enzymatic and structural analysis of BbhII reveals the architecture underlying its specificity and the presence of a GlcNAc-6S-specific carbohydrate-binding module (CBM) 32 with a distinct sugar recognition mode that B. bifidum takes advantage of to degrade mucin O-glycans. Comparative analysis of the genomes of prominent mucinolytic bacteria also highlights a CBM-dependent O-glycan breakdown strategy used by B. bifidum. [Figure not available: see fulltext.].

A fluorescence-quenching-based assay system to determine the hydrolytic activity of endo-β-N-acetylglucosaminidases (ENGases), which act on the innermost N-acetylglucosamine (GlcNAc) residue of the chitobiose segment of core-fucosylated N-glycans, was constructed using a dual-labeled fluorescent probe with a hexasaccharide structure. The fluorogenic probe was evaluated using a variety of ENGases, including Endo-M W251N mutant, Endo-F3, and Endo-S, which recognize core fucosylated N-glycans. The occurrence of a hydrolysis reaction was detected by observing an increased fluorescence intensity, ultimately allowing the ENGase activities to be easily and quantitatively evaluated, with the exception of Endo-S. The obtained results clearly indicated the substrate specificities of the examined ENGases.

Most functional biopharmaceuticals such as antibodies are glycoproteins carrying N-linked oligosaccharides (N-glycans). In animal cells, these glycans are generally expressed as heterogeneous glycoforms that are difficult to separate into a pure form. The structure of these glycans directly affects several biological aspects of the glycoproteins, especially binding affinity. Therefore, the preparation of glycoproteins with well-defined and homogeneous glycoforms is necessary for functional studies and improved efficacy, particularly for biopharmaceuticals. This review describes the recent remarkable progress in the development and production of biopharmaceutical glycan-modified antibodies, through the use of glycan remodeling using microbial endoglycosidases and sophisticated glycoengineering techniques utilizing microbial enzymatic reaction mechanisms.

Ovomucin, a hen egg white protein, is characterized by its hydrogel-forming properties, high molecular weight, and extensive O -glycosylation with a high degree of sialylation. As a commonly used food ingredient, we explored whether ovomucin has an effect on the gut microbiota. O- Glycan analysis revealed that ovomucin contained core-1 and 2 structures with heavy modification by N -acetylneuraminic acid and/or sulfate groups. Of the two mucin-degrading gut microbes we tested, Akkermansia muciniphila grew in medium containing ovomucin as a sole carbon source during a 24 h culture period, whereas Bifidobacterium bifidum did not. Both gut microbes, however, degraded ovomucin O -glycans and released monosaccharides into the culture supernatants in a species-dependent manner, as revealed by semi-quantified mass spectrometric analysis and anion exchange chromatography analysis. Our data suggest that ovomucin potentially affects the gut microbiota through O -glycan decomposition by gut microbes and degradant sugar sharing within the community.

Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO-assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose (FL)), are associated with the formation of a bifidobacteria-rich gut microbial community. Phylogenetic analysis has revealed that FL transporters have diversified into two clades harboring four clusters within the Bifidobacterium genus, but the underpinning functional diversity associated with this divergence remains underexplored. In this study, we examined the HMO-consumption phenotypes of two bifidobacterial species, Bifidobacterium catenulatum subspecies kashiwanohense and Bifidobacterium pseudocatenulatum, which both possess FL binding proteins that belong to phylogenetic clusters with unknown specificities. Growth assays, heterologous gene expression experiments, and HMO-consumption analysis showed that the FL transporter type from B. catenulatum subspecies kashiwanohense JCM 15439T conferred a novel HMO-uptake pattern that includes the complex fucosylated HMOs (lacto-N-fucopentaose II and lacto-N-difucohexaose I/II). Further genomic landscape analyses of FL transporter-positive bifidobacterial strains revealed that H-antigen or Lewis antigen-specific fucosidase gene(s) and FL transporter specificities were largely aligned. These results suggest that bifidobacteria have acquired FL transporters along with the corresponding gene sets necessary to utilize the imported HMOs. Our results provide insight into the species- and strain-dependent adaptation strategies of bifidobacteria to HMO-rich environments. Importance The gut of breastfed infants is generally dominated by health-promoting bifidobacteria. Human milk oligosaccharides (HMOs) from breastmilk selectively promote the growth of specific taxa such as bifidobacteria, thus forming an HMO-mediated, host-microbe symbiosis. While the co-evolution of humans and bifidobacteria has been proposed, the underpinning adaptive strategies employed by bifidobacteria require further research. Here, we analyzed the divergence of the critical fucosyllactose (FL) HMO transporter within Bifidobacterium. We have shown that the diversification of the solute-binding proteins of the FL-transporter led to uptake specificities of fucosylated sugars ranging from simple trisaccharides to complex hexasaccharides. This transporter and the congruent acquisition of the necessary intracellular enzymes allows for bifidobacteria to import different types of HMOs in a predictable and strain-dependent manner. These findings explain the adaptation and proliferation of bifidobacteria in the competitive and HMO-rich infant gut environment and enable accurate specificity annotation of transporters from metagenomic data.

The early life human gut microbiota exerts life-long health effects on the host, but the mechanisms underpinning its assembly remain elusive. Particularly, the early colonization of Clostridiales from the Roseburia-Eubacterium group, associated with protection from colorectal cancer, immune- and metabolic disorders is enigmatic. Here, we describe catabolic pathways that support the growth of Roseburia and Eubacterium members on distinct human milk oligosaccharides (HMOs). The HMO pathways, which include enzymes with a previously unknown structural fold and specificity, were upregulated together with additional glycan-utilization loci during growth on selected HMOs and in co-cultures with Akkermansia muciniphila on mucin, suggesting an additional role in enabling cross-feeding and access to mucin O-glycans. Analyses of 4599 Roseburia genomes underscored the preponderance and diversity of the HMO utilization loci within the genus. The catabolism of HMOs by butyrate-producing Clostridiales may contribute to the competitiveness of this group during the weaning-triggered maturation of the microbiota., The assembly and maturation of the early life microbiome has life-long effects on human health. Here, the authors combine omics, functional assays and structural analyses to characterize the catabolic pathways that support the growth of butyrate producing Clostridiales members from the Roseburia and Eubacterium, on distinct human milk oligosaccharides.

The gut microbiome is a dynamic community that significantly affects host health; it is frequently disturbed by medications such as antibiotics. Recently, probiotics have been proposed as a remedy for antibiotic-induced dysbiosis, but the efficacy of such treatments remains uncertain. Thus, the effect of specific antibiotic-probiotic combinations on the gut microbiome and host health warrants further research. We tested the effect vancomycin, amoxicillin, and ciprofloxacin on mice. Antibiotic administration was followed by one of the following recovery treatments: Bifidobacterium bifidum JCM 1254 as a probiotic (PR); fecal transplant (FT); or natural recovery (NR). Each antibiotic administration and recovery treatment was repeated three times over 9 weeks. We used the Shannon Index and Chao1 Index to determine gut microbiome diversity and assessed recovery by quantifying the magnitude of microbial shift using the Bray-Curtis Index of Dissimilarity. We determined the community composition by sequencing the V3–V4 regions of the 16S ribosomal RNA gene. To assess host health, we measured body weight and cecum weight, as well as mRNA expression of inflammation-related genes by reverse-transcription quantitative PCR. Our results show that community response varied by the type of antibiotic used, with vancomycin having the most significant effects. As a result, the effect of probiotics and fecal transplants also varied by antibiotic type. For vancomycin, the first antibiotic disturbance substantially increased the relative abundance of inflammatory species in the phylum Proteobacteria, such as Proteus, but the effect of subsequent disturbances was less pronounced, suggesting that the gut microbiome is affected by past disturbance events. Furthermore, although gut microbiome diversity did not recover, probiotic supplementation was effective in limiting cecum size enlargement and colonic inflammation caused by vancomycin. However, for amoxicillin and ciprofloxacin, the relative abundances of proinflammatory species were not greatly affected, and consequently, the effect of probiotic supplementation on community structure, cecum weight, and expression of inflammation-related genes was comparatively negligible. These results indicate that probiotic supplementation is effective, but only when antibiotics cause proinflammatory species-induced gut inflammation, suggesting that the necessity of probiotic supplementation is strongly influenced by the type of disturbance introduced to the community.

The early life human gut microbiota exerts life-long health effects on the host, but the mechanisms underpinning its assembly remain elusive. Particularly, the early colonization of Clostridiales from the Roseburia-Eubacterium group, associated with protection from colorectal cancer, immune- and metabolic disorders is enigmatic. Here we unveil the growth of Roseburia and Eubacterium members on human milk oligosaccharides (HMOs) using an unprecedented catabolic apparatus. The described HMO pathways and additional glycan utilization loci confer co-growth with Akkermansia muciniphilia via cross-feeding and access to mucin O-glycans. Strikingly, both, HMO and xylooligosaccharide pathways, were active simultaneously attesting an adaptation to a mixed HMO-solid food diet. Analyses of 4599 Roseburia genomes underscored the preponderance of HMO pathways and highlighted different HMO utilization phylotypes. Our revelations provide a possible rationale for the early establishment and resilience of butyrate producing Clostridiales and expand the role of specific HMOs in the assembly of the early life microbiota.

Aim: 16S rRNA gene-based microbiota analyses (16S metagenomes) using next-generation sequencing (NGS) technologies are widely used to examine the microbial community composition in environmental samples. However, the sequencing capacity of NGS is sometimes insufficient to cover the whole microbial community, especially when analyzing soil and fecal microbiotas. This limitation may have hampered the detection of minority species that potentially affect microbiota formation and structure. Methods: We developed a simple method, termed 16S metagenome-DRIP (Deeper Resolution using an Inhibitory Primer), that not only enhances minority species detection but also increases the accuracy of their abundance estimation. The method relies on the inhibition of normal amplicon formation of the 16S rRNA gene of a target major (abundant) species during the first PCR step. The addition of a biotinylated primer that is complementary to the variable sequence of the V3-V4 region of the target species inhibits a normal amplification process to form an aberrant short amplicon. The fragment is then captured by streptavidin beads for removal from the reaction mixture, and the resulting mixture is utilized for the second PCR with barcode-tag primers. Thus, this method only requires two additional experimental procedures to the conventional 16S metagenome analysis. A proof-of-concept experiment was first conducted using a mock sample consisting of the genomes of 14 bacterial species. Then, the method was applied to infant fecal samples using a Bifidobacterium-specific inhibitory primer (n = 11). Results: As a result, the reads assigned to the family Bifidobacteriaceae decreased on average from 16,657 to 1718 per sample without affecting the total read counts (36,073 and 34,778 per sample for the conventional and DRIP methods, respectively). Furthermore, the minority species detection rate increased with neither affecting Bray-Curtis dissimilarity calculated by omitting the target Bifidobacterium species (median: 0.049) nor changing the relative abundances of the non-target species. While 115 amplicon sequence variants (ASVs) were unique to the conventional method, 208 ASVs were uniquely detected for the DRIP method. Moreover, the abundance estimation for minority species became more accurate, as revealed thorough comparison with the results of quantitative PCR analysis. Conclusion: The 16S metagenome-DRIP method serves as a useful technique to grasp a deeper and more accurate microbiota composition when combined with conventional 16S metagenome analysis methods.

The chemo-enzymatic synthesis of an artificially N-glycosylated derivative of glucagon, a peptide hormone that regulates the blood sugar level, is described. We synthesized the glycosylated glucagon by chemical synthesis of an N-acetylglucosaminyl peptide and enzymatic transfer of an oligosaccharide using the transglycosylation activity of the glycosynthase-like mutant of Mucor hiemalis endo-β-N-acetylglucosaminidase (Endo-M) and sialo-oligosaccharide oxazoline as a donor substrate. The sialo-oligosaccharide-attached glucagon synthesized showed high resistance against protease degradation and stimulated the release of glucose from mouse hepatocytes when added to cells. The synthetic glucagon showed slightly higher activity than native glucagon and has potential as a therapeutic agent for treating diabetic patients.

We have recently generated a highly efficient 1,2-α-l-fucosynthase (BbAfcA N423H mutant) by protein engineering of 1,2-α-l-fucosidase from Bifidobacterium bifidum JCM 1254. This synthase could specifically introduce H-antigens (Fucα1-2Gal) into the non-reducing ends of oligosaccharides and in O-linked glycans in mucin glycoprotein. In the present study, we show an extended application of the engineered 1,2-α-l-fucosynthase by demonstrating its ability to insert Fuc residues into N- and O-glycans in fetuin glycoproteins, GM1 ganglioside, and a plant-derived xyloglucan nonasaccharide. This application study broadens the feasibility of this novel H-antigen synthesis technique in functional glycomics.

The human gut microbiota established during infancy has persistent effects on health. In vitro studies have suggested that human milk oligosaccharides (HMOs) in breast milk promote the formation of a bifidobacteria-rich microbiota in infant guts; however, the underlying molecular mechanism remains elusive. Here, we characterized two functionally distinct but overlapping fucosyllactose transporters (FL transporter-1 and -2) from Bifidobacterium longum subspecies infantis. Fecal DNA and HMO consumption analyses, combined with deposited metagenome data mining, revealed that FL transporter-2 is primarily associated with the bifidobacteria-rich microbiota formation in breast-fed infant guts. Structural analyses of the solute-binding protein (SBP) of FL transporter-2 complexed with 2′-fucosyllactose and 3-fucosyllactose, together with phylogenetic analysis of SBP homologs of both FL transporters, highlight a unique adaptation strategy of Bifidobacterium to HMOs, in which the gain-of-function mutations enable FL transporter-2 to efficiently capture major fucosylated HMOs. Our results provide a molecular insight into HMO-mediated symbiosis and coevolution between bifidobacteria and humans., ビフィズス菌におけるヒトへの適応進化を発見 --母乳オリゴ糖トランスポーターの獲得形質がビフィズスフローラ形成を促す--. 京都大学プレスリリース. 2019-09-05.

Endo-β-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-α1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic α1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glyco-oxazoline) onto an α1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics.

Fucα1-2 Gal linkages, or H-antigens, constitute histo-blood group antigens and are involved in various physiological processes. In addition, recent studies have shown that the H-antigen-containing glycans play an important role, not only in establishing harmonious relationship between gut microbes and the host, but also in preventing gut dysbiosis-related diseases. Therefore, development of an efficient method for introducing Fuc residue at Gal residue at the nonreducing end of glycans via α-(1→2) linkage is desired for research as well as medicinal purposes. In this study, we succeeded in derivatizing inverting 1,2-α-l-fucosidase (AfcA) into a highly efficient 1,2-α-l-fucosynthase. The synthase specifically synthesized H type 1-, type 2-, type 3- and type 4-chain-containing oligosaccharides with yields of 57-75% based on acceptor depletion. The synthase was also able to specifically introduce Fuc residues into Lewis a/x antigens to produce Lewis b/y antigens, with yields of 43% and 62%, respectively. In addition, the enzyme efficiently introduced H-antigens into sugar chains of porcine gastric mucins, as revealed by lectin blotting and mass spectroscopy analysis of the sugars. Detailed acceptor specificity analysis using various monosaccharides and oligosaccharides unraveled unique substrate recognition feature of this synthase at the subsite (+1), which can be explained by our previous X-ray crystallographic study of AfcA. These results show that the synthase developed in this study could serve as an alternative to other H-antigen synthesis methods involving α-1,2-fucosyltransferases and retaining α-fucosidase.

Certain species of the genus Bifidobacterium represent human symbionts. Many studies have shown that the establishment of symbiosis with such bifidobacterial species confers various beneficial effects on human health. Among the more than ten (sub)species of human gut-associated Bifidobacterium that have significantly varied genetic characteristics at the species level, Bifidobacterium bifidum is unique in that it is found in the intestines of a wide age group, ranging from infants to adults. This species is likely to have adapted to efficiently degrade host-derived carbohydrate chains, such as human milk oligosaccharides (HMOs) and mucin O-glycans, which enabled the longitudinal colonization of intestines. The ability of this species to assimilate various host glycans can be attributed to the possession of an adequate set of extracellular glycoside hydrolases (GHs). Importantly, the polypeptides of those glycosidases frequently contain carbohydrate-binding modules (CBMs) with deduced affinities to the target glycans, which is also a distinct characteristic of this species among members of human gut-associated bifidobacteria. This review firstly describes the prevalence and distribution of B. bifidum in the human gut and then explains the enzymatic machinery that B. bifidum has developed for host glycan degradation by referring to the functions of GHs and CBMs. Finally, we show the data of co-culture experiments using host-derived glycans as carbon sources, which underpin the interesting altruistic behavior of this species as a cross-feeder.

Sialidases catalyze the removal of terminal sialic acid from various complex carbohydrates. In the gastrointestinal tract, sialic acid is commonly found in the sugar chain of mucin, and many enteric commensals use mucin as a nutrient source. We previously identified two different sialidase genes in Bifidobacterium bifidum, and one was cloned and expressed as an extracellular protein designated as exo-α-sialidase SiaBb2. The other exo-α-sialidase gene (siabb1) from the same bifidobacterium encodes an extracellular protein (SiaBb1) consisting of 1795 amino acids with a molecular mass of 189 kDa. SiaBb1 possesses a catalytic domain that classifies this enzyme as a glycoside hydrolase family 33 member. SiaBb1 preferentially hydrolyzes α2,3-linked sialic acid over α2,6-linked sialic acid from sialoglycan, which is the same as SiaBb2. However, SiaBb1 has an SGNH hydrolase domain with sialate-O-acetylesterase activity and an N-terminal signal sequence and C-terminal transmembrane region. SiaBb1 is the first bifunctional sialidase identified with esterase activity. Abbreviations: GalNAc: N-acetyl-D-galactosamine; Fuc: L-fucose; Gal: D-galactose

Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic antibodies. While the biantennary complex-type oligosaccharide attached to Asn297 of the Fc is essential for antibody effector functions, fucose and outer-arm sugars attached to the core heptasaccharide that generate structural heterogeneity (glycoforms) exhibit unique biological activities. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for the development and quality control of therapeutic antibodies, and glycan profiles of the Fc are recognized as critical quality attributes. In the past decade our understanding of the influence of glycosylation on the structure/function of IgG-Fc has grown rapidly through X-ray crystallographic and nuclear magnetic resonance studies, which provides possibilities for the design of novel antibody therapeutics. Furthermore, the chemoenzymatic glycoengineering approach using endoglycosidase-based glycosynthases may facilitate the development of homogeneous IgG glycoforms with desirable functionality as next-generation therapeutic antibodies. Thus, the Fc glycans are fertile ground for the improvement of the safety, functionality, and efficacy of therapeutic IgG antibodies in the era of precision medicine.

Leuconostoc mesenteroides strain NTM048 has been shown to have intestinal IgA-inducing ability. In this study, we investigated the immunostimulant potency of an exopolysaccharide secreted from strain NTM048 (NTM048 EPS) in vitro and in vivo in a murine model. NTM048 EPS ranges in size from 10 to 40 kDa and is speculated to be mainly composed of glucose and fructose. The in vitro study revealed that NTM048 EPS induced total and antigen-specific IgA production by Peyer's patch cells and influenced Th1 and Th2 cell-mediated response in splenocytes. Oral administration of NTM048 EPS dose-dependently induced fecal IgA production accompanied by the up-regulation of retinoic acid synthase and transforming growth factor-β receptor genes in Peyer's patch cells. Flow cytometric analysis of the splenocytes revealed an increase of the CD3+ T-cell population and the ratio of CD4+ T-cells/CD8+ T-cells. These results indicate that NTM048 EPS could enhance the mucosal barrier and influence the systemic immune response.

We describe the novel substrate specificities of two independently evolved lacto-N-biosidases (LnbX and LnbB) towards the sugar chains of globo- and ganglio-series glycosphingolipids. LnbX, a non-classified member of the glycoside hydrolase family, isolated from Bifidobacterium longum subsp. longum, was shown to liberate galacto-N-biose (GNB: Galβ1-3GalNAc) and 2'-fucosyl GNB (a type-4 trisaccharide) from Gb5 pentasaccharide and globo H hexasaccharide, respectively. LnbB, a member of the glycoside hydrolase family 20 isolated from Bifidobacterium bifidum, was shown to release GNB from Gb5 and GA1 oligosaccharides. This is the first report describing enzymatic release of β-linked GNB from natural substrates. These unique activities may play a role in modulating the microbial composition in the gut ecosystem, and may serve as new tools for elucidating the functions of sugar chains of glycosphingolipids.

Protein O-mannosylation is a glycan modification that is required for normal nervous system development and function. Mutations in genes involved in protein O-mannosyl glycosylation give rise to a group of neurodevelopmental disorders known as congenital muscular dystrophies (CMDs) with associated CNS abnormalities. Our previous work demonstrated that receptor protein-tyrosine phosphatase ζ (RPTPζ)/phosphacan is hypoglycosylated in a mouse model of one of these CMDs, known as muscle-eye-brain disease, a disorder that is caused by loss of an enzyme (protein O-mannose β-1,2-N-acetylglucosaminyltransferase 1) that modifies O-mannosyl glycans. In addition, monoclonal antibodies Cat-315 and 3F8 were demonstrated to detect O-mannosyl glycan modifications on RPTPζ/phosphacan. Here, we show that O-mannosyl glycan epitopes recognized by these antibodies define biochemically distinct glycoforms of RPTPζ/phosphacan and that these glycoforms differentially decorate the surface of distinct populations of neural cells. To provide a further structural basis for immunochemically based glycoform differences, we characterized the O-linked glycan heterogeneity of RPTPζ/phosphacan in the early postnatal mouse brain by multidimensional mass spectrometry. Structural characterization of the O-linked glycans released from purified RPTPζ/phosphacan demonstrated that this protein is a significant substrate for protein O-mannosylation and led to the identification of several novel O-mannose-linked glycan structures, including sulfo-N-acetyllactosamine containing modifications. Taken together, our results suggest that specific glycan modifications may tailor the function of this protein to the unique needs of specific cells. Furthermore, their absence in CMDs suggests that hypoglycosylation of RPTPζ/phosphacan may have different functional consequences in neurons and glia.

Human gut symbiont bifidobacteria possess carbohydrate-degrading enzymes that act on the O-linked glycans of intestinal mucins to utilize those carbohydrates as carbon sources. However, our knowledge about mucin type O-glycan degradation by bifidobacteria remains fragmentary, especially regarding how they decompose sulfated glycans, which are abundantly found in mucin sugar-chains. Here, we examined the abilities of several Bifidobacterium strains to degrade a sulfated glycan substrate and identified a 6-sulfo-β-d-N-acetylglucosaminidase, also termed sulfoglycosidase, encoded by bbhII from Bifidobacterium bifidum JCM 7004. A recombinant BbhII protein showed a substrate preference toward 6-sulfated and 3,4-disulfated N-acetylglucosamines over non-sulfated and 3-sulfated N-acetylglucosamines. The purified BbhII directly released 6-sulfated N-acetylglucosamine from porcine gastric mucin and the expression of bbhII was moderately induced in the presence of mucin. This de-capping activity may promote utilization of sulfated glycans of mucin by other bacteria including bifidobacteria, thereby establishing the symbiotic relationship between human and gut microbes.

Recently, a “human gut microbial gene catalogue,” which ranks the dominance of microbe genus/species in human fecal samples, was published. Most of the bacteria ranked in the catalog are currently publicly available; however, the growth media recommended by the distributors vary among species, hampering physiological comparisons among the bacteria. To address this problem, we evaluated Gifu anaerobic medium (GAM) as a standard medium. Forty-four publicly available species of the top 56 species listed in the “human gut microbial gene catalogue” were cultured in GAM, and out of these, 32 (72%) were successfully cultured. Short-chain fatty acids from the bacterial culture supernatants were then quantified, and bacterial metabolic pathways were predicted based on in silico genomic sequence analysis. Our system provides a useful platform for assessing growth properties and analyzing metabolites of dominant human gut bacteria grown in GAM and supplemented with compounds of interest.

The exopolysaccharide (EPS) produced by probiotic Leuconostoc mesenteroides subsp. mesenteroides strain NTM048 has been reported to be an immunostimulant that enhances mucosal IgA production. In this study, we found that intranasal administration of mice with the EPS and an antigen (ovalbumin) resulted in secretion of antigen-specific IgA and IgG in the airway mucosa and the serum, suggesting that the EPS has the adjuvant activity for use with mucosal vaccination. Methylation analysis coupled to GC-MS, and 1D and 2D NMR spectroscopy revealed that 94% of the EPS consists of an α-(1 → 6) glucan containing 4% of 1→3-linked α-glucose branches. To determine structures of minor components, we enzymatically digested the glucan with dextranase and used 2D NMR spectroscopy to identify the remaining polymer as a fructan (or fructans), containing both β-(2 → 6)- and β-(2 → 1)-linked fructofuranose residues. These residues may either enter into separate polymers of each linkage type or form a mixed fructan containing both linkage types.

For chemo-enzymatic synthesis of a glycosylated peptide, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was used for the synthesis of a N-acetylglucosaminyl peptide and a pseudoglycopeptide by solid-phase peptide synthesis without the requirement of protecting groups on the carbohydrate. We also performed transglycosylation of an N-glycan to the N-acetylglucosaminyl peptide using endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) to synthesize a glycopeptide containing a complex N-glycan.

Endoplasmic reticulum (ER) α-glucosidase I is an enzyme that trims the distal α1,2-linked glucose (Glc) residue from the Glc3Man9GlcNAc2 oligosaccharide following its addition to nascent glycoproteins in the initial step of processing. This reaction is critical to the subsequent processing of N-glycans and thus defects in α-glucosidase I gene in human cause congenital disorder of glycosylation (CDG) type IIb. We identified the Caenorhabditis elegans α-glucosidase I gene (F13H10.4, designated agl-1) that encodes a polypeptide with 36% identity to human α-glucosidase I. The agl-1 cDNA restored the expression of complex-type N-glycans on the cell surface of α-glucosidase I-defective Chinese hamster ovary Lec23 cells. RNAi knockdown of agl-1 [agl-1(RNAi)] produced worms that were visibly similar to wild-type, but lifespan was reduced to about half of the control. Analyses of N-glycosylation in agl-1(RNAi) animals by western blotting and mass spectrometry showed reduction of paucimannose and complex-type glycans and dramatic increase of glucosylated oligomannose glycans. In addition, a significant amount of unusual terminally fucosylated N-glycans were found in agl-1(RNAi) animals. ER stress response was also provoked, leading to the accumulation of large amounts of triglucosylated free oligosaccharides (FOSs) (Glc3Man4–5GlcNAc1–2) in agl-1(RNAi) animals. Acceleration of ER-associated degradation in response to the accumulation of unfolded glycoproteins and insufficient interaction with calnexin/calreticulin in the ER lumen likely accounts for the increase of FOSs. Taken together, these studies in C. elegans demonstrate that decreased ER α-glucosidase I affects the entire N-glycan profile and induces chronic ER stress, which may contribute to the pathophysiology of CDG-IIb in humans.

Siglec-8 and Siglec-F are single pass transmembrane inhibitory receptors found on the surface of human and mouse eosinophils, respectively, but very little is known about their physiologic glycan ligands. This article reviews the latest knowledge on this topic and outlines the strategies being used to further define the production and glycobiochemical nature of these molecules in the lung.Both Siglec-8 and Siglec-F recognize the same glycan structure, namely 6'-sulfated sialyl Lewis X, as determined using glycan array technologies. Studies have identified α2,3-linked sialylated glycoprotein structures localized to mouse airway epithelium in tissue sections, where their constitutive expression requires the specific sialyltransferase St3gal3. Expression of these ligands in lung is enhanced during allergic inflammation and by cytokines such as IL-13, and is maintained in primary air-liquid interface cultures of mouse lung epithelium. Further characterization suggests that they are high molecular weight sialylated proteins, putatively mucins. By combining analytic glycomics, glycoproteomic mapping, and further in-vitro eosinophil experimentation including the ability of candidate structures to enhance eosinophil apoptosis, a finely detailed appreciation of the structural requirements for productive Siglec-8 and Siglec-F engagement should soon emerge.An enhanced understanding of Siglec-F, Siglec-8, and their ligands should improve our understanding of endogenous lung pathways limiting the survival of eosinophils within the airway in diseases such as asthma. Knowledge of this biology may also result in novel opportunities for drug development involving glycans and glycomimetics that selectively bind to Siglec-8 and induce eosinophil death.

The past 25 years have seen significant advances in understanding the diversity and functions of glycoprotein glycans in Drosophila melanogaster. Genetic screens have captured mutations that reveal important biological activities modulated by glycans, including protein folding and trafficking, as well as cell signaling, tissue morphogenesis, fertility, and viability. Many of these glycan functions have parallels in vertebrate development and disease, providing increasing opportunities to dissect pathologic mechanisms using Drosophila genetics. Advances in the sensitivity of structural analytic techniques have allowed the glycan profiles of wild-type and mutant tissues to be assessed, revealing novel glycan structures that may be functionally analogous to vertebrate glycans. This review describes a selected set of recent advances in understanding the functions of N-linked and O-linked (non-glycosaminoglycan) glycoprotein glycans in Drosophila with emphasis on their relatedness to vertebrate organisms.

A levansucrase from Leuconostoc mesenteroides NTM048 was cloned and expressed and its enzymatic product was characterized. The fructansucrase gene from Leuconostoc mesenteroides was cloned and expressed in Escherichia coli. The recombinant enzyme was purified as a single protein and its properties investigated. The polymer produced by the recombinant enzyme was identified as levan by various means including TLC and NMRs, and the enzyme was identified as a GH68 levansucrase. The enzyme was optimal at pH 5.5–6 and 30 °C, and its activity was stimulated by Ca2+. The levan produced by this strain induced IgA production in mice. Leuconostoc mesenteroides, a probiotic strain, possessed levansucrase which catalyzed the produced levan that had immunomodulating activity.

Glycan conversion of glycoprotein via the transglycosylation activity of endo-β-N-acetylglucosaminidase is a promising chemoenzymatic technology for the production of glycoproteins including bio-medicines with a homogeneous glycoform. Although Endo-M is a key enzyme in this process, its product undergoes rehydrolysis, which leads to a lower yield, and limits the practical application of this enzyme. We developed several Endo-M mutant enzymes including N175Q with glycosynthase-like activity and/or transglycosidase-like activity. We found that the Endo-M N175H mutant showed glycosynthase-like activity comparable to N175Q as well as transglycosidase-like activity superior to N175Q. Using a natural sialylglycopeptide as a donor substrate, N175H readily transferred the sialo-glycan onto an N-acetylglucosamine residue attached to bovine ribonuclease B (RNase B), yielding a nonnative sialoglycosylated RNase B. These results demonstrate that use of Endo-M N175H is an alternative glycoengineering technique, which provides a relatively high yield of transglycosylation product and avoids the laborious synthesis of a sugar oxazoline as a donor substrate.

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive β-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive β-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique for protein separation, and in-gel tryptic digestion of resolved protein bands has enhanced the resolution of protoeomic analysis. To augment this technology and expand its usefulness for glycoproteomics, we have developed and improved methods to release and recover O-linked glycans from proteins resolved in SDS-PAGE gels for subsequent analysis by mass spectrometry (MS). Gel pieces containing target proteins are washed to remove contaminants. O-linked glycans are released through reductive β-elimination by hydrating gel pieces in base and adding reductant. Following straightforward sample cleanup, this simple treatment of glycoprotein gel pieces produces material suitable for MS analysis. We have applied this method to the analysis of mucin-type glycoproteins that are expected to carry high densities of sialylated and sulfated O-linked glycans. However, the strongly acidic nature of the sulfate moiety suppresses MS signal intensities, hampering detection and quantitative analysis. To enhance detection, we present an improved method for sulfoglycomics. A mixture of sulflo-, sialo-, and neutral glycans were permethylated and partitioned into a water-dichloromethane (DCM) solvent mixture. Sulfated glycans were selectively recovered from the aqueous phase, while neutral and sialylated glycans remained in the DCM phase. When applied to the analysis of human mucin salivary glycans, this partition method generated material of sufficient quality to identify more than 60 glycan structures by NSI-MS (LTQ-Orbitrap) in positive and negative ion modes. Also, nearly 100% of the sulfated O-linked glycans were recovered in the aqueous phase, demonstrating the feasibility of in-depth sulfoglycomic analysis using SDS-PAGE resolved proteins.

Background Sialic acid–binding, immunoglobulin-like lectin (Siglec) F is a glycan-binding protein selectively expressed on mouse eosinophils. Its engagement induces apoptosis, suggesting a pathway for ameliorating eosinophilia in the setting of asthma and other eosinophil-associated diseases. Siglec-F recognizes sialylated sulfated glycans in glycan-binding assays, but the identities of endogenous sialoside ligands and their glycoprotein carriers in vivo are unknown. Objectives To use mouse lung-derived materials to isolate, biochemically identify, and biologically characterize naturally occurring endogenous glycan ligands for Siglec-F. Methods Lungs from normal and mucin-deficient mice, as well as mouse tracheal epithelial cells, were investigated in vitro and in vivo for the expression of Siglec-F ligands. Western blotting and cytochemistry used Siglec-F-Fc as a probe for directed purification, followed by liquid chromatography–tandem mass spectrometry of recognized glycoproteins. Purified components were tested in mouse eosinophil-binding assays and flow cytometry–based cell death assays. Results We detected mouse lung glycoproteins that bound to Siglec-F; binding was sialic acid dependent. Proteomic analysis of Siglec-F binding material identified Muc5b and Muc4. Cross-affinity enrichment and histochemical analysis of lungs from mucin-deficient mice assigned and validated the identity of Muc5b as one glycoprotein ligand for Siglec-F. Purified mucin preparations carried sialylated and sulfated glycans, bound to eosinophils and induced their death in vitro . Mice conditionally deficient in Muc5b displayed exaggerated eosinophilic inflammation in response to intratracheal installation of IL-13. Conclusions These data identify a previously unrecognized endogenous anti-inflammatory property of airway mucins by which their glycans can control lung eosinophilia through engagement of Siglec-F.