The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono-and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.

Endo-␤-N-acetylglucosaminidase from Mucor hiemalis (Endo-M), a family 85 glycoside hydrolase, acts on the ␤1,4 linkage of N,N-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glycoengineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low transglycosylation have hampered its broad applications. This paper describes the sitedirected mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the K m value of Y217F for an acceptor substrate 4-methylumbelliferyl-␤-D-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn 175 "knocked out" the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines (the transition state mimics) as donor substrates for transglycosylation. This is the first glycosynthase derived from endo-␤-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.Endo-␤-N-acetylglucosaminidase (EC 3.2.1.96) (ENGase) 3 catalyzes hydrolysis of the ␤1,4-glycosidic linkage of the N,NЈ-diacetylchitobiose moiety in the core of asparagine-linked glycan of various glycoproteins and glycopeptides. This type of enzyme is widely distributed in animals, plants, fungi, and bacteria. Several bacterial enzymes, such as Endo-H from Streptomyces plicatus (1) and Endo-F 1 from Flavobacterium meningosepticum (2), were cloned and classified into glycoside hydrolase (GH) family 18 in the CAZy data base (available on the World Wide Web), which may share a common evolutional origin with GH18 chitinases. The other ENGases are distinct from the enzymes of the GH18 chitinase family and are classified into the GH family 85. We and others have previously reported that several ENGases of the GH85 family showed significant transglycosylation activity (i.e. the ability to transfer the releasing glycan to an acceptor other than water to form a new glycosidic linkage) (3-6). These ENGases include Endo-M from Mucor hiemalis (3), Endo-A from Arthrobactor protophormiae (4), Endo-CE from Caenorhabditi...

The postsynaptic density (PSD) proteins Shank and Homer cooperate to induce the maturation and enlargement of dendritic spines (Sala et al., 2001). Homer1a is an activity-inducible short-splice variant of Homer that lacks dimerization capacity. Here, we show that Homer1a reduces the density and size of dendritic spines in cultured hippocampal neurons in correlation with an inhibition of Shank targeting to synapses. Expression of Homer1a also decreases the size of PSD-95 clusters, the number of NMDA receptor clusters, and the level of surface AMPA receptors, implying a negative effect on the growth of synapses. In parallel with the morphological effects on synapses, Homer1a-expressing neurons show diminished AMPA and NMDA receptor postsynaptic currents. All of these outcomes required the integrity of the Ena/VASP Homology 1 domain of Homer1a that mediates binding to the PPXXF motif in Shank and other binding partners. Overexpression of the C-terminal region of Shank containing the Homer binding site causes effects similar to those of Homer1a. These data indicate that an association between Shank and the constitutively expressed long-splice variants of Homer (e.g., Homer1b/c) is important for maintaining dendritic-spine structure and synaptic function. Because Homer1a expression is induced by synaptic activity, our results suggest that this splice variant of Homer operates in a negative feedback loop to regulate the structure and function of synapses in an activity-dependent manner.

Bifidobacteria are predominant bacteria present in the intestines of breast-fed infants and offer important health benefits for the host. Human milk oligosaccharides are one of the most important growth factors for bifidobacteria and are frequently fucosylated at their non-reducing termini. Previously, we identified 1,2-alpha-l-fucosidase (AfcA) belonging to the novel glycoside hydrolase (GH) family 95, from Bifidobacterium bifidum JCM1254 (Katayama T, Sakuma A, Kimura T, Makimura Y, Hiratake J, Sakata K, Yamanoi T, Kumagai H, Yamamoto K. 2004. Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-l-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J Bacteriol. 186:4885-4893). Here, we identified a gene encoding a novel 1,3-1,4-alpha-l-fucosidase from the same strain and termed it afcB. The afcB gene encodes a 1493-amino acid polypeptide containing an N-terminal signal sequence, a GH29 alpha-l-fucosidase domain, a carbohydrate binding module (CBM) 32 domain, a found-in-various-architectures (FIVAR) domain and a C-terminal transmembrane region, in this order. The recombinant enzyme was expressed in Escherichia coli and was characterized. The enzyme specifically released alpha1,3- and alpha1,4-linked fucosyl residues from 3-fucosyllactose, various Lewis blood group substances (a, b, x, and y types), and lacto-N-fucopentaose II and III. However, the enzyme did not act on glycoconjugates containing alpha1,2-fucosyl residue or on synthetic alpha-fucoside (p-nitrophenyl-alpha-l-fucoside). The afcA and afcB genes were introduced into the B. longum 105-A strain, which has no intrinsic alpha-l-fucosidase. The transformant carrying afcA could utilize 2'-fucosyllactose as the sole carbon source, whereas that carrying afcB was able to utilize 3-fucosyllactose and lacto-N-fucopentaose II. We suggest that AfcA and AfcB play essential roles in degrading alpha1,2- and alpha1,3/4-fucosylated milk oligosaccharides, respectively, and also glycoconjugates, in the gastrointestinal tracts.

A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the ␣-(132) linkage of 2-fucosyllactose, and a gene encoding 1,2-␣-L-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal ␣-(132)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2-fucosyllactose was determined to be inversion by using 1 H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).␣-L-Fucosyl residues are frequently found at the nonreducing termini of various glycoconjugates, including blood group substances, milk oligosaccharides, gastric and submaxillary mucins, and serum glycoproteins (30, 37). The results of recent studies indicate that such terminal fucosyl residues attached by

Homogeneous N-glycoproteins carrying defined natural N-glycans are essential for detailed structural and functional studies. The transglycosylation activity of the endo-β-N-acetylglucosaminidases from Arthrobacter protophormiae (Endo-A) and Mucor hiemalis (Endo-M) holds a great potential for glycoprotein synthesis, but the wild type enzymes are not practical for making glycoproteins carrying native N-glycans because of their predominant activity for product hydrolysis. We report in this article the studies on two endoglycosidase-based glycosynthases, EndoM-N175A and EndoA-N171A, and their usefulness for constructing homogeneous N-glycoproteins carrying natural N-glycans. Oligosaccharide oxazoline corresponding to the bi-antennary complex type N-glycan was synthesized and tested with the two glycosynthases. The EndoM-N175A mutant was able to efficiently transfer the complex type glycan oxazoline to a GlcNAc-peptide and GlcNAc-containing ribonuclease to form the corresponding homogeneous glycopeptide/glycoprotein. The EndoA-N171A did not recognize complex type N-glycan oxazoline but could efficient use the high-mannose type glycan oxazoline for transglycosylation. These mutants possess the transglycosylation activity but lack the hydrolytic activity toward the product. Kinetic studies revealed that the dramatically enhanced synthetic efficiency of the EndoA-N171A mutant was due to the significantly reduced hydrolytic activity toward both the Man9GlcNAc oxazoline and the product, as well as its enhanced activity for transglycosylation. Thus, the two mutants described here represent the first endoglycosidase-based glycosynthases enabling a high efficient synthesis of homogeneous natural N-glycoproteins.

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 (Gal␤1,3GlcNAc; type 1 chain) from lacto-N-tetraose (Gal␤1,3GlcNAc␤1,3Gal␤1,4Glc), 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 ␤-linked lacto-Nbiose 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.

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