Description: In four yeast strains, Ogataea minuta , Candida parapolymorpha , Pichia anomala and Zygosaccharomyces rouxii , we identified endo-β- N -acetylglucosaminidase (ENGase) homologous sequences by database searches; in each of the four species, a corresponding enzyme activity was also confirmed in crude cell extract obtained from each strain. The O. minuta ENGase (Endo-Om)-encoding gene was directly amplified from O. minuta genomic DNA and sequenced. The Endo-Om-encoding gene contained a 2319-bp open-reading frame; the deduced amino acid sequence indicated that the putative protein belonged to glycoside hydrolase family 85. The gene was introduced into O. minuta , and the recombinant Endo-Om was overexpressed and purified. When the enzyme assay was performed using an agalacto-biantennary oligosaccharide as a substrate, Endo-Om exhibited both hydrolysis and transglycosylation activities. Endo-Om exhibited hydrolytic activity for high-mannose, hybrid, biantennary and (2,6)-branched triantennary N-linked oligosaccharides, but not for tetraantennary, (2,4)-branched triantennary, bisecting N -acetylglucosamine structure and core-fucosylated biantennary N-linked oligosaccharides. Endo-Om also was able to hydrolyze N-glycans attached to RNase B and human transferrin under both denaturing and nondenaturing conditions. Thus, the present study reports the detection and characterization of a novel yeast ENGase.

Description: Human milk oligosaccharides contain a large variety of oligosaccharides, of which lacto-N-biose I (Gal-β1,3-GlcNAc; LNB) predominates as a major core structure. A unique metabolic pathway specific for LNB has recently been identified in the human commensal bifidobacteria. Several strains of infant gut-associated bifidobacteria possess lacto-N-biosidase, a membrane-anchored extracellular enzyme, that liberates LNB from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds. Lacto-N-biosidase belongs to the glycoside hydrolase family 20, and its reaction proceeds via a substrate-assisted catalytic mechanism. Several crystal structures of GH20 β-N-acetylhexosaminidases, which release monosaccharide GlcNAc from its substrate, have been determined, but to date, a structure of lacto-N-biosidase is unknown. Here, we have determined the first three-dimensional structures of lacto-N-biosidase from Bifidobacterium bifidum JCM1254 in complex with LNB and LNB-thiazoline (Gal-β1,3-GlcNAc-thiazoline) at 1.8-Å resolution. Lacto-N-biosidase consists of three domains, and the C-terminal domain has a unique β-trefoil-like fold. Compared with other β-N-acetylhexosaminidases, lacto-N-biosidase has a wide substrate-binding pocket with a −2 subsite specific for β-1,3-linked Gal, and the residues responsible for Gal recognition were identified. The bound ligands are recognized by extensive hydrogen bonds at all of their hydroxyls consistent with the enzyme's strict substrate specificity for the LNB moiety. The GlcNAc sugar ring of LNB is in a distorted conformation near 4E, whereas that of LNB-thiazoline is in a 4C1 conformation. A possible conformational pathway for the lacto-N-biosidase reaction is discussed.

Description: When nucleotide-binding oligomerization domain–like receptors (NLRs) sense cytosolic-invading bacteria, they induce the formation of inflammasomes and initiate an innate immune response. In quiescent cells, inflammasome activity is tightly regulated to prevent excess inflammation and cell death. Many bacterial pathogens provoke inflammasome activity and induce inflammatory responses, including cell death, by...

Description: Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1–3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase+ strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1–3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc), and sialyllacto-N-tetraose a (Neu5Acα2–3Galβ1–3GlcNAcβ1–3Galβ1–4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1–3GlcNAcβ-pNP) and GalNAcβ1–3GlcNAcβ-pNP. GalNAcβ1–3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca2+ and Mg2+) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.

Description: Endoplasmic reticulum (ER) α-glucosidase I is an enzyme that trims the distal α1,2-linked glucose (Glc) residue from the Glc 3 Man 9 GlcNAc 2 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) (Glc 3 Man 4–5 GlcNAc 1–2 ) 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.

Description: APIP, Apaf-1 interacting protein, has been known to inhibit two main types of programmed cell death, apoptosis and pyroptosis, and was recently found to be associated with cancers and inflammatory diseases. Distinct from its inhibitory role in cell death, APIP was also shown to act as a 5-methylthioribulose-1-phosphate dehydratase, or...

Description: 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.

Description: Due to dramatic advances in DNA technology, quantitative measures of annotation data can now be obtained in continuous coordinates across the entire genome, allowing various heterogeneous ‘genomic landscapes’ to emerge. Although much effort has been devoted to comparing DNA sequences, not much attention has been given to comparing these large quantities of data comprehensively. In this article, we introduce a method for rapidly detecting local regions that show high correlations between genomic landscapes. We overcame the size problem for genome-wide data by converting the data into series of symbols and then carrying out sequence alignment. We also decomposed the oscillation of the landscape data into different frequency bands before analysis, since the real genomic landscape is a mixture of embedded and confounded biological processes working at different scales in the cell nucleus. To verify the usefulness and generality of our method, we applied our approach to well investigated landscapes from the human genome, including several histone modifications. Furthermore, by applying our method to over 20 genomic landscapes in human and 12 in mouse, we found that DNA replication timing and the density of Alu insertions are highly correlated genome-wide in both species, even though the Alu elements have amplified independently in the two genomes. To our knowledge, this is the first method to align genomic landscapes at multiple scales according to their shape.

Description: Bifidobacterium bifidum is one of the most frequently found bifidobacteria in the intestines of newborn infants. We previously reported that B. bifidum possesses unique metabolic pathways for O -linked glycans on gastrointestinal mucin (Yoshida E, Sakurama H, Kiyohara M, Nakajima M, Kitaoka M, Ashida H, Hirose J, Katayama T, Yamamoto K, Kumagai H. 2012. Bifidobacterium longum subsp. infantis uses two different β-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides. Glycobiology . 22:361–368). The nonreducing termini of O -linked glycans on mucin are frequently covered with histo-blood group antigens. Here, we identified a gene agabb from B. bifidum JCM 1254, which encodes glycoside hydrolase (GH) family 110 α-galactosidase. AgaBb is a 1289-amino acid polypeptide containing an N-terminal signal sequence, a GH110 domain, a carbohydrate-binding module (CBM) 51 domain, a bacterial Ig-like (Big) 2 domain and a C-terminal transmembrane region, in this order. The recombinant enzyme expressed in Escherichia coli hydrolyzed α1,3-linked Gal in branched blood group B antigen [Galα1-3(Fucα1-2)Galβ1-R], but not in a linear xenotransplantation antigen (Galα1-3Galβ1-R). The enzyme also acted on group B human salivary mucin and erythrocytes. We also revealed that CBM51 specifically bound blood group B antigen using both isothermal titration calorimetry and a solid-phase binding assay, and it enhanced the affinity of the enzyme toward substrates with multivalent B antigens. We suggest that this enzyme plays an important role in degrading B antigens to acquire nutrients from mucin oligosaccharides in the gastrointestinal tracts.

Description: We examined the plasma flow response to meso- and microscale magnetic dipoles by performing three-dimensional full particle-in-cell simulations. We particularly focused on the formation of a magnetosphere and its dependence on the intensity of the magnetic moment. The size of a magnetic dipole immersed in a plasma flow can be characterized by a distance L from the dipole center to the position where the pressure of the local magnetic field becomes equal to the dynamic pressure of the plasma flow under the magnetohydrodynamics (MHD) approximation. In this study, we are interested in a magnetic dipole whose L is smaller than the Larmor radius of ions r iL calculated with the unperturbed dipole field at the distance L from the center. In the simulation results, we confirmed the clear formation of a magnetosphere consisting of a magnetopause and a tail region in the density profile, although the spatial scale is much smaller than the MHD scale. One of the important findings in this study is that the spatial profiles of the plasma density as well as the current flows are remarkably affected by the finite Larmor radius effect of the plasma flow, which is different from the Earth's magnetosphere. The magnetopause found in the upstream region is located at a position much closer to the dipole center than L . In the equatorial plane, we also found an asymmetric density profile with respect to the plasma flow direction, which is caused by plasma gyration in the dipole field region. The ion current layers are created in the inner region of the dipole field, and the electron current also flows in the region beyond the ion current layer because ions with a large inertia can closely approach the dipole center. Unlike the ring current structure of the Earth's magnetosphere, the current layers in the microscale dipole fields are not circularly closed around the dipole center. Since the major current is caused by the particle gyrations, the current is independently determined to be in the direction of the electron and ion gyrations, which are the same in both the upstream and downstream regions. The present analysis on the formation of a magnetosphere in the regime of a microscale magnetic dipole is significant for understanding the solar wind response to the crustal magnetic anomalies on the Moon surface, such as were recently observed by spacecraft.