Neonatal Exposure to Commensal-Bacteria-Derived Antigens Directs Polysaccharide-Specific B-1 B Cell Repertoire Development.

B-1 B cells derive from a developmental program distinct from that of conventional B cells, through B cell receptor (BCR)-dependent positive selection of fetally derived precursors. Here, we used direct labeling of B cells reactive with the N-acetyl-D-glucosamine (GlcNAc)-containing Lancefield group A carbohydrate of Streptococcus pyogenes to study the effects of bacterial antigens on the emergent B-1 B cell clonal repertoire. The number, phenotype, and BCR clonotypes of GlcNAc-reactive B-1 B cells were modulated by neonatal exposure to heat-killed S. pyogenes bacteria. GlcNAc-reactive B-1 clonotypes and serum antibodies were reduced in germ-free mice compared with conventionally raised mice. Colonization of germ-free mice with a conventional microbiota promoted GlcNAc-reactive B-1 B cell development and concomitantly elicited clonally related IgA+ plasma cells in the small intestine. Thus, exposure to microbial antigens in early life determines the clonality of the mature B-1 B cell repertoire and ensuing antibody responses, with implications for vaccination approaches and schedules.

Escherichia coli BL21(DE3) optimized deletion mutant as the host for whole-cell biotransformation of N­acetyl­D­neuraminic acid.

N­Acetyl­D­neuraminic acid (Neu5Ac) is the crucial compound for the chemical synthesis of antiflu medicine Zanamivir. Chemoenzymatic synthesis of Neu5Ac involves N-acetyl-D-glucosamine 2-epimerase (AGE)-catalyzed epimerization of N-acetyl-D-glucosamine (GlcNAc) to N-acetyl-D-mannosamine (ManNAc), and aldolase-catalyzed condensation between ManNAc and pyruvate. Host optimization plays an important role in the whole-cell biotransformation of value-added compounds. In this study, via single-plasmid biotransformation system, we showed that the AGE gene BT0453, cloned from human gut microorganism Bacteroides thetaiotaomicron VPI-5482, showed the highest biotransformation yield among the AGE genes tested; and there is no clear Neu5Ac yield difference between the BT0453 coupled with one aldolase coding nanA gene and two nanA genes. Next, Escherichia coli chromosomal genes involved in substrate degradation, product exportation and pH change were deleted via recombineering and CRISPR/Cas9. With the final E. coli BL21(DE3) ΔnanA Δnag ΔpoxB as host, a significant 16.5% yield improvement was obtained. Furthermore, precursor (pyruvate) feeding resulted in 3.2% yield improvement, reaching 66.8% molar biotransformation. The result highlights the importance of host optimization, and set the stage for further metabolic engineering of whole-cell biotransformation of Neu5Ac.

Unraveling the Impact of Acetylation Patterns in Chitosan Oligomers on Cu2+ Ion Binding: Insights from DFT Calculations.

Chitosans are partially acetylated polymers of glucosamine, structurally characterized by their degree of polymerization as well as their fraction and pattern of acetylation. These parameters strongly influence the physico-chemical properties and biological activities of chitosans, but structure-function relationships are only poorly understood. As an example, we here investigated the influence of acetylation on chitosan-copper complexation using density functional theory. We investigated the electronic structures of completely deacetylated and partially acetylated chitosan oligomers and their copper-bound complexes. Frontier molecular orbital theory revealed bonding orbitals for electrophiles and antibonding orbitals for nucleophiles in fully deacetylated glucosamine oligomers, while partially acetylated oligomers displayed bonding orbitals for both electrophiles and nucleophiles. Our calculations showed that the presence of an acetylated subunit in a chitosan oligomer affects the structural and the electronic properties of the oligomer by generating new intramolecular interactions with the free amino group of neighboring deacetylated subunits, thereby influencing its polarity. Furthermore, the band gap energy calculated from the fully and partially deacetylated oligomers indicates that the mobility of electrons in partially acetylated chitosan oligomers is higher than in fully deacetylated oligomers. In addition, fully deacetylated oligomers form more stable complexes with higher bond dissociation energies with copper than partially acetylated ones. Interestingly, in partially acetylated oligomers, the strength of copper binding was found to be dependent on the pattern of acetylation. Our study provides first insight into the influence of patterns of acetylation on the electronic and ion binding properties of chitosans. Depending on the intended application, the obtained results can serve as a guide for the selection of the optimal chitosan for a specific purpose.

Functional Characterization of the GlcNAc Catabolic Pathway in Cryptococcus deneoformans.

The amino sugar N-acetyl-d-glucosamine (GlcNAc) is the key constituent of cell wall components and plays an important role in pathogenesis in a wide range of fungi. However, catabolism of GlcNAc has not been studied in basidiomycete fungi. In this study, we identified and characterized a gene cluster essential for GlcNAc utilization in Cryptococcus deneoformans, an environmental human fungal pathogen. The C. deneoformans genome contains a GlcNAc transporter (Ngt1), a GlcNAc kinase (Hxk3), a GlcNAc-6-phosphate deacetylase (Dac1), and a glucosamine-6-phosphate deaminase (Nag1). Their expression levels were highly induced in cultures containing GlcNAc as the sole carbon source, and the corresponding mutants showed severe growth defects in the presence of GlcNAc. Functional and biochemical analyses revealed that HXK3 encodes a novel GlcNAc kinase. Site-directed mutations of conserved residues of Hxk3 indicated that ATP binding and GlcNAc binding are essential for GlcNAc kinase activities. Taken together, the results from this study provide crucial insights into basidiomycete GlcNAc catabolism. IMPORTANCEN-Acetylglucosamine (GlcNAc) is recognized as not only the building block of chitin but also an important signaling molecule in fungi. The catabolic pathway of GlcNAc also plays an important role in vital biological processes in fungi. However, the utilization pathway of GlcNAc in the phylum Basidiomycota, which contains more than 41,000 species, remains unknown. Cryptococcus deneoformans is a representative basidiomycetous pathogen that causes life-threatening meningitis. In this study, we characterized a gene cluster essential for GlcNAc utilization in C. deneoformans and identified a novel GlcNAc kinase. The results of this study provide important insights into basidiomycete GlcNAc catabolism and offer a starting point for revealing its role in pathogenesis.

Protecting-group- and microwave-free synthesis of ß-glycosyl esters and aryl ß-glycosides of N-acetyl-d-glucosamine.

A protecting-group-free method for synthesis of ß-glycosyl esters and aryl ß-glycosides was developed by using latent chemical reactivity of N-acetyl-d-glucosamine (GlcNAc) oxazoline. The GlcNAc oxazoline was spontaneously reacted with carboxylic acids and phenol derivatives via the oxazoline ring opening without the use of a catalyst or heating conditions (i.e., microwave irradiation), affording the desired products in moderate to excellent yields with ß-selectivity. This simple protecting-group-free method exhibits a wide substrate scope and good functional group tolerance, and it allows the efficient production of a novel class of GlcNAc-conjugated biomaterials and prodrug candidates.

Efficient production of GlcNAc in an aqueous-organic system with a Chitinolyticbacter meiyuanensis SYBC-H1 mutant.

OBJECTIVES: Shellfish waste is a primary source for making N-acetyl-D-glucosamine. Thus, establishing a high-efficiency and low-cost bioconversion method to produce N-acetyl-D-glucosamine directly from shellfish waste was promising. RESULTS: A mutant C81 was obtained from Chitinolyticbacter meiyuanensis SYBC-H1 via 60Co-γ irradiation. This mutant C81 showed the highest chitinase activity of 9.8 U/mL that was 85% higher than the parent strain. The mutant C81 exhibted improved antioxidant activities, including total antioxidant capacity, superoxide radical ability, and hydroxyl radical scavenging ability, compared to that of the parent strain. Four out of nine organic solvents increased the chitinase activity by 1.9%, 6.8%, 11.7%, and 15.8%, corresponding to methylbenzene, n-heptane, petroleum ether, and n-hexane, respectively. The biphase system composed of aqueous and hexane presented a five-fold reduction of cell viability compared to the control. Using a continuous fermentation bioconversion process, 4.2 g/L GlcNAc was produced from crayfish shell powder with a yield of 80% of the chitin content. CONCLUSIONS: This study demonstrated that the mutant C81 is suitable for converting crayfish shell powder into GlcNAc in an aqueous-organic system.

Recent advances in the bioprospection and applications of chitinolytic bacteria for valorization of waste chitin.

One of the most abundant natural polymers on earth, chitin is a fibrous and structural polysaccharide, composed of N-acetyl-D-glucosamine. The biopolymer is the major structural constituent of fungi, arthropods, mollusks, nematodes, and some algae. The biodegradation of chitin is largely manifested by chitinolytic enzyme secreting organisms including bacteria, insects, and plants. Among them, bacterial chitinases represent the most promising, inexpensive, and sustainable source of proteins that can be employed for industrial-scale applications. To this end, the presented review comes at a timely moment to highlight the major sources of chitinolytic bacteria. It also discusses the potential pros and cons of prospecting bacterial chitinases that can be easily manipulated through genetic engineering. Additionally, we have elaborated the recent applications of the chitin thereby branding chitinases as potential candidates for biorefinery and biomedical research for eco-friendly and sustainable management of chitin waste in the environment.

Nitration of Chitin Monomer: From Glucosamine to Energetic Compound.

The nitration of chitin monomer in a mixture of nitric acid and acetic anhydride was conducted and a highly nitrated (3R,4R,6R)-3-acetamido-6-((nitrooxy)methyl)tetrahydro-2H-pyran-2,4,5-triyl trinitrate (1) was obtained. Its structure was fully characterized using infrared spectroscopy, NMR spectroscopy, elemental analysis, and X-ray diffraction. Compound 1 possesses good density (ρ: 1.721 g·cm-3) and has comparable detonation performance (Vd: 7717 m·s-1; P: 25.6 GPa) to that of nitrocellulose (NC: Vd: 7456 m·s-1; P: 23 GPa; Isp = 239 s) and microcrystalline nitrocellulose (MCNC; Vd: 7683 m·s-1; P: 25 GPa; Isp = 250 s). However, Compound 1 has much lower impact sensitivity (IS: 15 J) than the regular nitrocellulose (NC; IS: 3.2 J) and MCNC (IS: 2.8 J). Compound 1 was calculated to exhibit a good specific impulse (Isp: 240 s), which is comparable with NC (Isp: 239 s) and MCNC (Isp: 250 s). By replacing the nitrocellulose with Compound 1 in typical propellants JA2, M30, and M9, the specific impulse was improved by up to 4 s. These promising properties indicate that Compound 1 has a significant potential as an energetic component in solid propellants.

Characterization of mouse di-N-acetylchitobiase that can degrade chitin-oligosaccharides.

Di-N-acetylchitobiase (Ctbs) degrades ß-1,4 glycoside bonds of the chitobiose core of free asparagine-linked glycan. This study examined whether Ctbs degrades chitin-oligosaccharides to GlcNAc in mammals. We analyzed Ctbs mRNA and protein expression in mouse tissues and characterized enzymatic activity using recombinant mouse Ctbs expressed in Escherichia coli. Ctbs mRNA and protein were expressed in various tissues of mouse, including the stomach. Optimal conditions for recombinant Ctbs were pH 3.0 and 45°C, and the recombinant enzyme was retained more than 94% activity after incubation at pH 3.0-7.0 and below 37°C. The recombinant Ctbs hydrolyzed (GlcNAc)3 and (GlcNAc)6 at pH 3.0 and produced GlcNAc. The K m of Ctbs was lowest with (GlcNAc)3 as a substrate. k cat/K m was fourfold as high with (GlcNAc)3 and (GlcNAc)4 as substrates than with (GlcNAc)2. These results suggest that Ctbs digests chitin-oligosaccharides or (GlcNAc)2 of reducing-end residues of oligosaccharides and produces GlcNAc in mouse tissues.

Structural basis for antibody targeting of the broadly expressed microbial polysaccharide poly-N-acetylglucosamine.

In response to the widespread emergence of antibiotic-resistant microbes, new therapeutic agents are required for many human pathogens. A non-mammalian polysaccharide, poly-N-acetyl-d-glucosamine (PNAG), is produced by bacteria, fungi, and protozoan parasites. Antibodies that bind to PNAG and its deacetylated form (dPNAG) exhibit promising in vitro and in vivo activities against many microbes. A human IgG1 mAb (F598) that binds both PNAG and dPNAG has opsonic and protective activities against multiple microbial pathogens and is undergoing preclinical and clinical assessments as a broad-spectrum antimicrobial therapy. Here, to understand how F598 targets PNAG, we determined crystal structures of the unliganded F598 antigen-binding fragment (Fab) and its complexes with N-acetyl-d-glucosamine (GlcNAc) and a PNAG oligosaccharide. We found that F598 recognizes PNAG through a large groove-shaped binding site that traverses the entire light- and heavy-chain interface and accommodates at least five GlcNAc residues. The Fab-GlcNAc complex revealed a deep binding pocket in which the monosaccharide and a core GlcNAc of the oligosaccharide were almost identically positioned, suggesting an anchored binding mechanism of PNAG by F598. The Fab used in our structural analyses retained binding to PNAG on the surface of an antibiotic-resistant, biofilm-forming strain of Staphylococcus aureus Additionally, a model of intact F598 binding to two pentasaccharide epitopes indicates that the Fab arms can span at least 40 GlcNAc residues on an extended PNAG chain. Our findings unravel the structural basis for F598 binding to PNAG on microbial surfaces and biofilms.

Development of an Escherichia coli-based biocatalytic system for the efficient synthesis of N-acetyl-D-neuraminic acid.

N-acetyl-d-neuraminic acid (Neu5Ac) is a valuable resource that has seen increasing demand in both medicine and biotechnology. Although enzymatic systems and whole-cell biocatalysts have been developed for the synthesis of Neu5Ac, low yield and productivity still hamper the use of these methods on larger scales. We report the creation of an Escherichia coli biocatalyst for the efficient synthesis of Neu5Ac using a metabolic and protein engineering strategy. Expression of the two enzymes, N-acetyl-D-glucosamine 2-epimerase (AGE) and Neu5Ac lyase (NAL), was balanced using promoter engineering. Genes encoding competing pathways and GlcNAc catabolism were deleted, and then a structure-guided process was used to identify a more efficient NAL and an AGE mutant with a higher rate of Neu5Ac synthesis. The resulting biocatalyst produced 351.8 mM Neu5Ac with a yield of 58.6% from GlcNAc. This work exemplifies the use of rational design and protein engineering to construct a complex bacterial biocatalyst that can serve as a platform for the large-scale synthesis of a useful biological material.

Identification and characterization of chitinolytic bacteria isolated from a freshwater lake.

To develop a novel type of biocontrol agent, we focus on bacteria that are characterized by both chitinase activity and biofilm development. Chitinolytic bacteria were isolated from sediments and chitin flakes immersed in the water of a sand dune lake, Sakata, in Niigata, Japan. Thirty-one isolates from more than 5100 isolated strains were examined chitinase activity and biofilm formation. Phylogenetic analysis of these isolates based on the 16S rRNA gene sequences revealed that most isolates belonged to the family Aeromonadaceae, followed by Paenibacillaceae, Enterobacteriaceae, and Neisseriaceae. The specific activity of chitinase of four selected strains was higher than that of a reference strain. The molecular size of one chitinase produced by Andreprevotia was greater than that of typical bacterial chitinases. The dialyzed culture supernatant containing chitinases of the four strains suppressed hyphal growth of Trichoderma reesei. These results indicate that these four strains are good candidates for biocontrol agents.

Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract.

Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.

Dietary Natural N-Acetyl-d-Glucosamine Prevents Bone Loss in Ovariectomized Rat Model of Postmenopausal Osteoporosis.

Postmenopausal osteoporosis has seriously affected the life quality of elderly women. A natural polymer, chitin, obtained from shells of crab and shrimp, has been widely used in the biomedical field owing to its nontoxicity, biocompatibility, and biodegradability. In this study, natural N-acetyl-d-glucosamine (NAG) was prepared from liquefied chitin. The protective activities of NAG in postmenopausal osteoporosis were evaluated on Sprague Dawley rats and osteoblast-based models. Results showed that oral administration of NAG boosted trabecular bone volume and trabecular numbers. Additionally, the calcium content in the femur and tibia increased, and femoral biomechanical properties improved. Furthermore, NAG supplementation significantly lowered alkaline phosphatase levels and increased calcium content in the serum of ovariectomized rats. In vitro studies showed that NAG markedly promoted cell proliferation and stimulated osteoblast differentiation of mouse calvaria origin MC3T3-E1 cells with increased alkaline phosphatase activity in a concentration-dependent manner. Moreover, NAG effectively protected osteoblasts from oxidative damage induced by hydrogen peroxide. In conclusion, our data provide an additional foundation for dietary supplementation of NAG, which could protect and reverse osteopenia in postmenopausal women.

Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane.

The partially de-N-acetylated poly-ß-1,6-N-acetyl-d-glucosamine (dPNAG) polymer serves as an intercellular biofilm adhesin that plays an essential role for the development and maintenance of integrity of biofilms of diverse bacterial species. Translocation of dPNAG across the bacterial outer membrane is mediated by a tetratricopeptide repeat-containing outer membrane protein, PgaA. To understand the molecular basis of dPNAG translocation, we determined the crystal structure of the C-terminal transmembrane domain of PgaA (residues 513-807). The structure reveals that PgaA forms a 16-strand transmembrane ß-barrel, closed by four loops on the extracellular surface. Half of the interior surface of the barrel that lies parallel to the translocation pathway is electronegative, suggesting that the corresponding negatively charged residues may assist the secretion of the positively charged dPNAG polymer. In vivo complementation assays in a pgaA deletion bacterial strain showed that a cluster of negatively charged residues proximal to the periplasm is necessary for biofilm formation. Biochemical analyses further revealed that the tetratricopeptide repeat domain of PgaA binds directly to the N-deacetylase PgaB and is critical for biofilm formation. Our studies support a model in which the positively charged PgaB-bound dPNAG polymer is delivered to PgaA through the PgaA-PgaB interaction and is further targeted to the ß-barrel lumen of PgaA potentially via a charge complementarity mechanism, thus priming the translocation of dPNAG across the bacterial outer membrane.

Development and characterization of new and scalable topical formulations containing N-acetyl-d-glucosamine-loaded solid lipid nanoparticles.

N-Acetyl-d-glucosamine (NAG) has been recently considered for topical treatment of hyperpigmentation disorders due to its inhibitory effect on thyrosinase enzymes in melanocytes. NAG is a precursor of hyaluronic acid, increasing its amount in skin, and consequently, preserving the skin hydration and elasticity. It may also act as an emulsion stabilizer. Solid lipid nanoparticles (SLN) are advanced delivery systems successfully used in pharmaceutical and cosmetic formulations for the improvement of active molecules penetration into the skin. Therefore, this work aimed to develop and characterize stable and scalable topical formulations containing NAG-loaded SLN. NAG was incorporated in SLN which were prepared by two high shear homogenizers and characterized regarding its morphology and particle size by transmission electron microscopy and photon correlation spectroscopy, respectively. Oil emulgel and hydrogel were used as carriers of NAG-loaded SLN. Several parameters were evaluated, including the droplet size distribution, rheology, pH and topical delivery by different techniques. It was observed that SLN size was significantly dependent on NAG incorporation and homogenization process. Most tested SLN parameters appeared to be quite suitable, that is, spherical and well-defined SLN with approximately 258 nm and -30 mV. Hereafter, both gels containing SLN presented a pseudoplastic flow. Emulgel formulation containing NAG-loaded SLN allowed a higher NAG permeation through the SC compared to the respective control (about 0.8 µgcm-2 h-1). According to the results obtained, it can be suggested that NAG acts as an emulsion stabilizer. This stabilization was also particularly dependent on the homogenizer type which is quite important for scale-up process. This study demonstrated the potential of scalable SLN formulations to improve NAG topical delivery contributing to the improvement of skin properties on several skin disorders.

Effect of incubation atmosphere on the production and composition of staphylococcal biofilms.

Staphylococcus aureus and Staphylococcus epidermidis are pathogenic bacteria that often cause invasive infections in humans. In this study, we characterized the composition and growth characteristics of staphylococcal biofilms under various incubation atmospheres. We assessed the effect of incubation atmosphere (aerobic, 5% CO2, anaerobic, and microaerobic) on the biofilm production capabilities of S. aureus strains isolated from healthy volunteers and from patients with catheter-related bloodstream infection. In addition, the composition of S. aureus and S. epidermidis biofilms was determined by assessment of biofilm degradation after treatment with DNase I, proteinase K, and dispersin B. The strains obtained from healthy volunteers and patients showed similar biofilm formation capabilities. Biofilms of S. aureus were rich in proteins when developed under ambient atmospheric conditions, 5% CO2, and microaerobic condition, whereas S. epidermidis biofilms contained large amounts of poly-ß (1, 6)-N-acetyl-D-glucosamine when developed under ambient atmospheric conditions and microaerobic condition. The biofilm-producing capability of S. epidermidis was considerably higher than that of S. aureus under aerobic condition. Staphylococcal isolates obtained from healthy individuals and patients with catheter-related infections have similar biofilm-forming capabilities. Under microaerobic conditions, S. aureus and S. epidermidis form protein-rich and poly-ß (1, 6)-N-acetyl-D-glucosamine-rich biofilms, respectively. These components may play an important role in the development of biofilms inside the body and may be the target molecules to prevent catheter-related infections caused by these organisms.

Metabolomic Analysis of Blood Plasma after Oral Administration of N-acetyl-d-Glucosamine in Dogs.

N-acetyl-d-glucosamine (GlcNAc) is a monosaccharide that polymerizes linearly through (1,4)-ß-linkages. GlcNAc is the monomeric unit of the polymer chitin. GlcNAc is a basic component of hyaluronic acid and keratin sulfate found on the cell surface. The aim of this study was to examine amino acid metabolism after oral GlcNAc administration in dogs. Results showed that plasma levels of ectoine were significantly higher after oral administration of GlcNAc than prior to administration (p < 0.001). To our knowledge, there have been no reports of increased ectoine concentrations in the plasma. The mechanism by which GlcNAc administration leads to increased ectoine plasma concentration remains unclear; future studies are required to clarify this mechanism.

The Autonomous Glycosylation of Large DNA Viruses.

Glycosylation of surface molecules is a key feature of several eukaryotic viruses, which use the host endoplasmic reticulum/Golgi apparatus to add carbohydrates to their nascent glycoproteins. In recent years, a newly discovered group of eukaryotic viruses, belonging to the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) group, was shown to have several features that are typical of cellular organisms, including the presence of components of the glycosylation machinery. Starting from initial observations with the chlorovirus PBCV-1, enzymes for glycan biosynthesis have been later identified in other viruses; in particular in members of the Mimiviridae family. They include both the glycosyltransferases and other carbohydrate-modifying enzymes and the pathways for the biosynthesis of the rare monosaccharides that are found in the viral glycan structures. These findings, together with genome analysis of the newly-identified giant DNA viruses, indicate that the presence of glycogenes is widespread in several NCLDV families. The identification of autonomous viral glycosylation machinery leads to many questions about the origin of these pathways, the mechanisms of glycan production, and eventually their function in the viral replication cycle. The scope of this review is to highlight some of the recent results that have been obtained on the glycosylation systems of the large DNA viruses, with a special focus on the enzymes involved in nucleotide-sugar production.

Discovery of ß-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase involved in the metabolism of N-glycans.

A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, ß-N-acetylhexosaminidase, exo-α-sialidase, and endo-ß-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of ß-1,4-D-mannosyl-N-acetyl-D-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where ß-1,4-D-mannosyl-N-acetyl-D-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-D-mannose 1-phosphate and N-acetyl-D-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where ß-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the D-mannose residue of ß-1,4-D-mannosyl-N-acetyl-D-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-ß-D-mannopyranosyl-N-acetyl-D-glucosamine:phosphate α-D-mannosyltransferase as the systematic name and ß-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase as the short name for BT1033.