Masashi Kiyohara

Bifidobacterium bifidum Lacto-N-Biosidase, a Critical Enzyme for the Degradation of Human Milk Oligosaccharides with a Type 1 Structure

ABSTRACT Breast-fed infants often have intestinal microbiota dominated by bifidobacteria in contrast to formula-fed infants. We found that several bifidobacterial strains produce a lacto-N-biosidase that liberates lacto-N-biose I (Galβ1,3GlcNAc; type 1 chain) from lacto-N-tetraose (Galβ1,3GlcNAcβ1,3Galβ1,4Glc), which is a major component of human milk oligosaccharides, and subsequently isolated the gene from Bifidobacterium bifidum JCM1254. The gene, designated lnbB, was predicted to encode a protein of 1,112 amino acid residues containing a signal peptide and a membrane anchor at the N and C termini, respectively, and to possess the domain of glycoside hydrolase family 20, carbohydrate binding module 32, and bacterial immunoglobulin-like domain 2, in that order, from the N terminus. The recombinant enzyme showed substrate preference for the unmodified β-linked lacto-N-biose I structure. Lacto-N-biosidase activity was found in several bifidobacterial strains, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli, under the tested conditions. These results, together with our recent finding of a novel metabolic pathway specific for lacto-N-biose I in bifidobacterial cells, suggest that some of the bifidobacterial strains are highly adapted for utilizing human milk oligosaccharides with a type 1 chain.

Cooperation of β-galactosidase and β-N-acetylhexosaminidase from bifidobacteria in assimilation of human milk oligosaccharides with type 2 structure

Bifidobacteria are predominant in the intestines of breast-fed infants and offer health benefits to the host. Human milk oligosaccharides (HMOs) are considered to be one of the most important growth factors for intestinal bifidobacteria. HMOs contain two major structures of core tetrasaccharide: lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc; type 1 chain) and lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc; type 2 chain). We previously identified the unique metabolic pathway for lacto-N-tetraose in Bifidobacterium bifidum. Here, we clarified the degradation pathway for lacto-N-neotetraose in the same bifidobacteria. We cloned one β-galactosidase (BbgIII) and two β-N-acetylhexosaminidases (BbhI and BbhII), all of which are extracellular membrane-bound enzymes. The recombinant BbgIII hydrolyzed lacto-N-neotetraose into Gal and lacto-N-triose II, and furthermore the recombinant BbhI, but not BbhII, catalyzed the hydrolysis of lacto-N-triose II to GlcNAc and lactose. Since BbgIII and BbhI were highly specific for lacto-N-neotetraose and lacto-N-triose II, respectively, they may play essential roles in degrading the type 2 oligosaccharides in HMOs.

An exo-α-sialidase from bifidobacteria involved in the degradation of sialyloligosaccharides in human milk and intestinal glycoconjugates

Bifidobacteria are health-promoting enteric commensals that are assumed to proliferate predominantly in the intestines of breast-fed infants by assimilating human milk oligosaccharides (HMOs) that are frequently fucosylated and/or sialylated. We previously identified two different α-l-fucosidases in Bifidobacterium bifidum and showed that the strain furnishes an extracellular degradation pathway for fucosylated HMOs. However, the catabolism of sialylated HMOs by bifidobacteria has remained unresolved. Here we describe the identification and characterization of an exo-α-sialidase in bifidobacteria. By expression cloning, we isolated a novel exo-α-sialidase gene (siabb2) from B. bifidum JCM1254, which encodes a protein (SiaBb2) consisting of 835-amino-acid residues with a predicted molecular mass of 87 kDa. SiaBb2 possesses an N-terminal signal sequence, a sialidase catalytic domain classified into the glycoside hydrolase family 33 (GH33) and a C-terminal transmembrane region, indicating that the mature SiaBb2 is an extracellular membrane-anchored enzyme. The recombinant enzyme expressed in Escherichia coli showed the highest activity in an acidic pH range from 4.0 to 5.0, and at 50 °C. Notably, 80% activity remained after 30 min incubation at 80 °C, indicating that the enzyme is highly thermostable. SiaBb2 liberated sialic acids from sialyloligosaccharides, gangliosides, glycoproteins and colominic acid; however, the linkage preference of the enzyme was remarkably biased toward the α2,3-linkage rather than α2,6- and α2,8-linkages. Expression of siabb2 in B. longum 105-A, which has no endogenous exo-α-sialidase, enabled this strain to degrade sialyloligosaccharides present in human milk. Our results suggest that SiaBb2 plays a crucial role in bifidobacterial catabolism of sialylated HMOs.

Bifidobacterium longum subsp. infantis uses two different β-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides

The breast-fed infant intestine is often colonized by particular bifidobacteria, and human milk oligosaccharides (HMOs) are considered to be bifidogenic. Recent studies showed that Bifidobacterium longum subsp. infantis can grow on HMOs as the sole carbon source. This ability has been ascribed to the presence of a gene cluster (HMO cluster-1) contained in its genome. However, the metabolism of HMOs by the organism remains unresolved because no enzymatic studies have been completed. In the present study, we characterized β-galactosidases of this subspecies to understand how the organism degrades type-1 (Galβ1-3GlcNAc) and type-2 (Galβ1-4GlcNAc) isomers of HMOs. The results revealed that the locus tag Blon_2016 gene, which is distantly located from the HMO cluster-1, encodes a novel β-galactosidase (Bga42A) with a significantly higher specificity for lacto-N-tetraose (LNT; Galβ1-3GlcNAcβ1-3Galβ1-4Glc) than for lacto-N-biose I (Galβ1-3GlcNAc), lactose (Lac) and type-2 HMOs. The proposed name of Bga42A is LNT β-1,3-galactosidase. The Blon_2334 gene (Bga2A) located within the HMO cluster-1 encodes a β-galactosidase specific for Lac and type-2 HMOs. Real-time quantitative reverse transcription-polymerase chain reaction analysis revealed the physiological significance of Bga42A and Bga2A in HMO metabolism. The organism therefore uses two different β-galactosidases to selectively degrade type-1 and type-2 HMOs. Despite the quite rare occurrence in nature of β-galactosidases acting on type-1 chains, the close homologs of Bga42A were present in the genomes of infant-gut associated bifidobacteria that are known to consume LNT. The predominance of type-1 chains in HMOs and the conservation of Bga42A homologs suggest the coevolution of these bifidobacteria with humans.

α-N-Acetylgalactosaminidase from Infant-associated Bifidobacteria Belonging to Novel Glycoside Hydrolase Family 129 Is Implicated in Alternative Mucin Degradation Pathway

Background: The degradation pathway of the intestinal mucin by bifidobacteria is poorly understood. Results: A novel α-N-acetylgalactosaminidase, NagBb, was identified from Bifidobacterium bifidum JCM 1254. Conclusion: NagBb might be involved in intracellular degradation of Tn antigen (GalNAcα1-Ser/Thr). Significance: NagBb represents a novel glycoside hydrolase family 129 in the CAZy database. Bifidobacteria inhabit the lower intestine of mammals including humans where the mucin gel layer forms a space for commensal bacteria. We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo-α-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. However, EngBF is highly specific for core-1-type O-glycan (Galβ1–3GalNAcα1-Ser/Thr), also called T antigen, which is mainly attached onto gastroduodenal mucins. By contrast, core-3-type O-glycans (GlcNAcβ1–3GalNAcα1-Ser/Thr) are predominantly found on the mucins in the intestines. Here, we identified a novel α-N-acetylgalactosaminidase (NagBb) from Bifidobacterium bifidum JCM 1254 that hydrolyzes the Tn antigen (GalNAcα1-Ser/Thr). Sialyl and galactosyl core-3 (Galβ1–3/4GlcNAcβ1–3(Neu5Acα2–6)GalNAcα1-Ser/Thr), a major tetrasaccharide structure on MUC2 mucin primarily secreted from goblet cells in human sigmoid colon, can be serially hydrolyzed into Tn antigen by previously identified bifidobacterial extracellular glycosidases such as α-sialidase (SiaBb2), lacto-N-biosidase (LnbB), β-galactosidase (BbgIII), and β-N-acetylhexosaminidases (BbhI and BbhII). Because NagBb is an intracellular enzyme without an N-terminal secretion signal sequence, it is likely involved in intracellular degradation and assimilation of Tn antigen-containing polypeptides, which might be incorporated through unknown transporters. Thus, bifidobacteria possess two distinct pathways for assimilation of O-glycans on gastroduodenal and intestinal mucins. NagBb homologs are conserved in infant-associated bifidobacteria, suggesting a significant role for their adaptation within the infant gut, and they were found to form a new glycoside hydrolase family 129.

Prebiotic Effect of Lacto-N-biose I on Bifidobacterial Growth

We demonstrated the prebiotic effect of lacto-N-biose I (Galβ1-3GlcNAc) on bifidobacteria in vitro. Lacto-N-biose I, a building unit of the type-I milk oligosaccharides, enhanced the growth of many bifidobacteria, especially Bifidobacterium bifidum, B. breve, and B. longum, which are predominant in the intestines of breast-fed infants. It might be a substantial, natural prebiotic in human colostrums.

Characterization of two different endo-α-N-acetylgalactosaminidases from probiotic and pathogenic enterobacteria, Bifidobacterium longum and Clostridium perfringens

Endo-alpha-N-acetylgalactosaminidase (endo-alpha-GalNAc-ase) catalyzes the hydrolysis of the O-glycosidic bond between alpha-GalNAc at the reducing end of mucin-type sugar chains and serine/threonine of proteins to release oligosaccharides. Previously, we identified the gene engBF encoding endo-alpha-GalNAc-ase from Bifidobacterium longum, which specifically released the disaccharide Gal beta 1-3GalNAc (Fujita K, Oura F, Nagamine N, Katayama T, Hiratake J, Sakata K, Kumagai H, Yamamoto K. 2005. Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum. J Biol Chem. 280:37415-37422). Here we cloned a similar gene named engCP from Clostridium perfringens, a pathogenic enterobacterium, and characterized the gene product EngCP. Detailed analyses on substrate specificities of EngCP and EngBF using a series of p-nitrophenyl-alpha-glycosides chemically synthesized by the di-tert-butylsilylene-directed method revealed that both enzymes released Hex/HexNAc beta 1-3GalNAc (Hex = Gal or Glc). EngCP could also release the core 2 trisaccharide Gal beta 1-3(GlcNAc beta 1-6)GalNAc, core 8 disaccharide Gal alpha 1-3GalNAc, and monosaccharide GalNAc. Our results suggest that EngCP possesses broader substrate specificity than EngBF. Actions of the two enzymes on native glycoproteins and cell surface glycoproteins were also investigated.

A Novel Endoglycoceramidase Hydrolyzes Oligogalactosylceramides to Produce Galactooligosaccharides and Ceramides

Enzymes capable of hydrolyzing the β-glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids has been found in microorganisms and invertebrates and designated endoglycoceramidase (EC 3.2.1.123) or ceramide glycanase. Here we report the molecular cloning, characterization, and homology modeling of a novel endoglycoceramidase that hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides. The novel enzyme was purified from a culture supernatant of Rhodococcus equi, and the gene encoding 488 deduced amino acids was cloned using peptide sequences of the purified enzyme. Eight residues essential for the catalytic reaction in microbial and animal endoglycoceramidases were all conserved in the deduced amino acid sequence of the novel enzyme. Homology modeling of the enzyme using endocellulase E1 as a template revealed that the enzyme displays a (β/α)8 barrel structure in which Glu234 at the end of β-strand 4 and Glu341 at the end of β-strand 7 could function as an acid/base catalyst and a nucleophile, respectively. Site-directed mutagenesis of these glutamates resulted in a complete loss of the activity without a change in their CD spectra. The recombinant enzyme hydrolyzed the β-galactosidic linkage between oligosaccharides and ceramides of 6-gala series glycosphingolipids that were completely resistant to hydrolysis by the enzymes reported so far. In contrast, the novel enzyme did not hydrolyze ganglio-, globo-, or lactoseries glycosphingolipids. The enzyme is therefore systematically named “oligogalactosyl-N-acylsphingosine 1,1′-β-galactohydrolase” or tentatively designated “endogalactosylceramidase.”

Changes in Microbiota Population during Fermentation of Narezushi as Revealed by Pyrosequencing Analysis

Modern Japanese sushi is derived from an archetype, narezushi, which is made by the fermentation of salted fish with rice. Several studies have demonstrated that lactic acid bacteria are dominantly present in narezushi, but no studies have addressed how microbial composition changes during fermentation. In this study, we examined changes in the microbial population in aji (horse mackerel)-narezushi during fermentation by pyrosequencing the 16S ribosomal RNA gene (rDNA). Ribosomal Database Project Classifier analysis revealed that among the 53 genera present, the Lactobacillus population drastically increased during fermentation, while the populations of other bacteria remained unchanged. Basic Local Alignment Search Tool analysis revealed that L. plantarum and L. brevis were the major species. Comparison with other fermented food microbiota indicated high product-dependency of the bacterial composition, which might have been due to the starter-free fermentation process.

Lacto-N-biosidase encoded by a novel gene of Bifidobacterium longum subspecies longum shows unique substrate specificity and requires a designated chaperone for its active expression.

Background: Phenotypically lacto-N-biosidase-positive Bifidobacterium longum JCM1217 does not possess a gene homologous to previously identified lacto-N-biosidase. Results: Hypothetical proteins BLLJ_1505 and BLLJ_1506 encode lacto-N-biosidase and its designated chaperone, respectively. Conclusion: The enzyme showed unique and unexpected substrate specificity. Significance: The enzyme is important for understanding how B. longum consumes human milk oligosaccharides and also may serve as a new tool in glycobiology. 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.